Systems and methods to quantify and amplify both signaling probes for cDNA chips and genes expression microarrays

ABSTRACT

The invention provides a series of reagent compositions and methods for making and amplifying novel cDNA based probe sets from RNA samples to improve analysis with gene expression arrays. The methods globally produce probe sets with common universal linkers at one or both ends, called WRAP-Probes, wherein the linkers do not bind to the target sequences and they can efficiently bind added reporters to the probes. The universal linkers are also designed as primer binding sites for copying and amplifying the probes, either linearly with one linker, or exponentially with double linkers. The capacity to globally and exponentially amplify the probe set by PCR is a primary advantage. Adding reporters by terminal linkers also improves quantification since each probe gets equivalent signaling. The invention allows expression analysis of small research, clinical and forensic samples to enable improved diagnostics, drug discovery, therapeutic monitoring, and medical, agricultural and general research.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims the benefit, pursuant to 35 U.S.C. §119(e), ofapplicant's provisional U.S. patent application Ser. No. 60/187,982,filed Mar. 9, 2000, entitled “Methods to Quantify and Amplify BothSignaling and Probes for DNA Chips and Gene Expression Microarrays”,which is hereby incorporate by reference herein for all purposes. Thisapplication further is a continuation-in-part of, and claims thebenefit, pursuant to 35 U.S.C. §120, of, applicant's U.S. patentapplication Ser. No. 09/744,097 filed Jan. 16, 2001 entitled “Methodsfor Detecting and Mapping Genes, Mutations and Variant PolynucleotideSequences,” which is hereby incorporated by reference herein for allpurposes and which is a National Stage Application of InternationalPatent Application Serial No. PCT/US99/16242 filed Jul. 16, 1999.

BACKGROUND OF INVENTION

1. Field of Invention

The present invention relates generally to the field of detecting genesand gene expression from biological and medical samples and moreparticularly it relates to improving both sensitivity and quantificationin comparative multi-analyte detection formats such as cDNA chips andexpression microarrays.

2. Description of Related Art

Genetic analysis of an organism or tissue involves two major fields ofstudy, the determination of existing genes and mutations as reflected ingenomic DNA sequences and the evaluation of functional gene activity asreflected in the expression of messenger RNA (mRNA) transcripts orresulting protein byproducts. Since there are no reasonable means toseparately detect all or most protein products simultaneously, globalcomparisons of gene expression have generally focused on mRNA analysisbecause such transcripts can be isolated and detected more simply—eitherby virtue of their specific sequences and or by virtue of the commonpresence of a poly-A tail on their 3′ end. These poly-A tails allow theentire pool of mRNAs to be simultaneously copied with a single poly-Tprimer and the enzyme reverse transcriptase (RT) to make a singleantisense strand of cDNA from each mRNA transcript in a sample.Consequently, most methods for gene expression analysis have primarilybeen based on assessing the relative number of RNA transcripts beingproduced by different genes and on comparing the timing of such geneactivity. The most important goal of these methods is therefore todetermine the comparative frequency of each transcript in differentcells and tissues, as well as detecting any expression changes thatoccur in response to various stimuli, physiological conditions andpathologic states. Furthermore, such quantitative methods should havebroad utility for genetics research in general and for a variety ofbiomedical applications including tissue typing and forensic analysis,the diagnosis and prognosis of various pathologies, conditions, andresponses to therapy, and the identification of new or refined targetsfor pharmaceutical therapy or gene therapy.

Current art has provided few methods to globally explore gene expressiondifferences between cells and tissues and most studies have employeddifferential display or cDNA subtraction analysis which provide partialnon-quantitative information [Hedrick et al., Nature 308: 149 (1984);Liang et. al., Science 257: 967, (1992)]. Similarly, expression analysisby Northern blotting, RNase protection assays, or reverse transcriptasepolymerase chain reaction (RT-PCR) are generally only useful forevaluating a very limited number of genes per analysis [Alwine, et al.,Proc. Natl. Acad. Sci., 74: 5350, (1977); Zinn, et al., Cell, 34: 865(1983); Veres, et al., Science, 237: 415 (1987)]. Several methods havebeen devised to extract cDNA copies of the 3′ ends of mRNA transcriptsand then characterize those fragments by restriction digests [Ivanovaet. al., Nucleic Acids Res. 23: 2954 (1995); Prashar et. al., Proc.Natl. Acad. Sci., 93: 659 (1996); Kato, Nucleic Acids Res. 23: 3685(1995); Kato, U.S. Pat. No. 5,707,807 (1998); Weissman et al., U.S. Pat.No. 5,712,126 (1998)]. While these methods expand the number ofexpression products that can be studied, they also remain limited inscope. Taking a different approach, Kinzler, et al. [U.S. Pat. No.5,695,937 (1998)] have devised a more comprehensive method for measuringmessenger RNA (mRNA) transcripts quantitatively by extracting andslicing out a tiny segment of the cDNAs copied from the 3′ end of eachmRNA transcript and then creating composite concatemers of thosesegments from different transcripts. The representative 9 or 10 basesegments are then counted by sequencing analysis to determine thefrequency of the original transcripts. However, this method involvesconsiderable complexity and the sequencing steps are very time consumingand expensive.

The development of cDNA based gene expression microarrays provides aready means to simultaneously assess the relative expression of hundredsor thousands of different genes from tissue or cellular samples. [Schenaet al., Science, 270: 467-470 (1995); Schena, et al., Proc. Natl. Acad.Sci., 93:10614-9 (1996); Shalon et al., Genome Res., 6: 639-45 (1996);DeRisi et al., Nature Genetics, 14: 457-60, (1996); Heller et al., Proc.Natl. Acad. Sci., 94: 2150-5, (1997); Khan et al., Cancer Res., 58:5009-13 (1998); Khan et al., Electrophoresis, 20: 223-9 (1999)] Theseanalyses are accomplished by first preparing miniature grids or arrayson membranes or coated glass substrates wherein small but dense cDNAsamples of individual genes are robotically spotted in a two dimensionalpattern. Then, a total RNA or mRNA sample is copied and labeled usingreverse transcriptase and a poly-T primer to create a pool of cDNAprobes that reflect the mRNA expression transcripts. These labeledprobes are then hybridized to their respective gene spots in themicroarray in order to detect and determine the relative frequency ofeach transcript in the original sample. These gene expression arrays,which are commonly called expression microarrays, DNA chips, cDNA chips,or biochips, were first manufactured from gene specific syntheticoligonucleotides that likewise are created or distributed on the arrayin a two dimensional pattern and that can capture and detect labeledexpression products in a somewhat similar manner if they are fragmentedinto smaller pieces [Fodor et al., U.S. Pat. No. 5,445,934 (1995); Fodoret al., U.S. Pat. No. 5,800,992 (1998)]. These commercial oligo-basedDNA chips are called GENECHIPS. It should be noted that microarraysgenerally refer to miniature arrays on coated glass substrates, however,larger scale arrays on membrane formats employ similar chemistries andtarget configurations and thus are suitable for and similarly improvedby the application of the present invention.

While the development of expression microarrays allows a greatlyexpanded overview and assessment of the relative frequency of differentgene transcripts in a sample, current methods are limited by significantdeficiencies in both quantification and sensitivity [Duggan et al.,Nature Genetics, 21: 10-14 (1999); DiRisi et al., Nature Genetics, 14:457-460 (1996); Rajeevan et al., Jour. Histochem. Cytochem., 47: 337-42(1999)]. Firstly, quantification is falsely biased since labeling isproportional to probe length, and thus, short genes give less signalingper probe than long genes. Secondly, even long genes provide limitedsignaling with cDNA chips when compared to the signaling provided by thefar longer segments that are typically used for mapping genes tochromosomes or nuclei. In addition, labeling is also limited forexpression microarrays because fluorescent compounds, such as Cy3 andCy5, which are commonly employed for comparative two color labeling, arepoorly incorporated by reverse transcriptase. Moreover, current methodsare especially limited in sensitivity when individual genes of interesthave been down-regulated or are weakly expressed or when the totalsample available for study is quite small. In either case, specific ormultiple gene transcripts of interest may produce an insufficient numberof labeled probes to be detected. Thus, current cDNA chip methods aregenerally poor or inadequate for detecting specific mRNA transcriptsthat are expressed in frequencies of less than 10 copies per cell or foranalyzing samples comprised of: a) less than 0.5 milligrams of tissue,b) less than 50 micrograms of total RNA, b) less than 0.5 micrograms ofpoly-A mRNA, or c) less than 5 million cells [Duggan et al., NatureGenetics, 21: 10-14 (1999)]. The conjunction of these deficiencies inboth quantification and sensitivity additionally creates furtherproblems. Thus, short genes may falsely appear inactive or weaklyexpressed relative to longer genes in the same sample, and longer geneswill falsely appear to be expressed more abundantly relative to shortergenes. Consequently, more accurate and sensitive detection methods areneeded.

One approach to improve chip detection would be to amplify mRNA derivedprobes by the polymerase chain reaction (PCR) or related enzymaticmethods. However, commonly available PCR procedures such as RT-PCR andmultiplex PCR, have only been used successfully to amplify a limitednumber of the gene products in a sample since effective multi-analyteamplification typically requires the provision of at least one uniqueprimer for each type of gene product amplified [Sutcliffe et al., U.S.Pat. No. 5,807,680 (1998)]. In related art such as differential displayor other older procedures to explore expression differences, globalamplification methods have been employed based upon using simplearbitrary primers, hexamers or various random primer constructs insteadof unique primers to amplify DNA or RNA. The inconsistency of suchmethods, however, have only made them useful for identifying unusual ornovel gene expression products, and they have not been devised oremployed for use with expression microarrays or DNA chip analyses [Welshet al., Nucleic Acids Res., 18: 7213-18 (1990); Pardee et. al., U.S.Pat. Nos. 5,262,311 (1993) and 5,665,547 (1997); Liang et al., NucleicAcids Res., 21: 3269 (1993); Mou et al., Biochem. Biophys. Res. Comm.,199: 564-569 (1994); Villeponteau et al., U.S. Pat. No. 5,580,726,(1996); Silver et. al., U.S. Pat. No. 5,104,792 (1992); Tavtigian etal., U.S. Pat. No. 5,789,206 (1998); Shuber, U.S. Pat. No. 5,882,856(1999)]. The prime difficulty with many of these methods derives fromthe use of short arbitrary or random primers that can give variableresults with different temperature and hybridization conditions suchthat they are unsuitable for diagnostic analyses. Even RT-PCR ormultiplex PCR methods, which employ unique primers, can producesemi-quantitative rather than quantitative results since differentprimer sets vary considerably in efficiency and since kinetic factorsfavor copying the smaller and more abundant products with those methods.Therefore, some products may not amplify well, and rare ordown-regulated transcripts may be under-represented [Khan et al.,Electrophoresis, 20: 223-9 (1999)]. Additionally, mammalian mRNA samplesinclude very large gene transcripts 6 to 12 thousand nucleotides longthat cannot be amplified reliably by routine PCR methods. Consequently,global PCR amplification of a pool of mRNA-derived cDNA probes has notbeen attempted or successfully accomplished for DNA chip or expressionmicroarray analyses, and based on the above reasons, it has beenscientific dogma that exponential amplification methods cannot bevalidly applied to multi-analyte gene expression arrays. Nonetheless,less robust linear amplification has been developed and employed forchip analyses by adding a RNA polymerase promoter to the end of thepoly-T primer used for RT. However, such amplification is incrementaland finite, with a typical duplication of 20-60 copies, and theamplified products it produces are antisense RNAs which are degradable[Phillips et. al., Methods, 10: 283-288 (1996); Kondo et al., U.S. Pat.No. 5,972,607; VanGelder et al., U.S. Pat. No. 5,716,785 (1998)]. Inrelated art, Wang et al., [U.S. Pat. No. 5,932,451 (1999)] refined suchmethods to allow asymmetrical PCR amplification of ds cDNA made from anmRNA sample. However, this amplification method is similarly limited inthe number of copies that can reasonably be made from the originalsample (68 fold duplication demonstrated). More importantly, by copyingfull length probes, the signaling bias of current methods cannot beovercome since the number of labels incorporated per probe is a largevariable dictated by the transcript size of different genes, and incommon mammalian species including humans, transcripts vary from severalhundred bases to twelve thousand bases or more. These problems thereforesuggested that improved detection might be better achieved by amplifyingsignaling rather than the target sample.

As described in PCT/US99/16242 (WIPO Publication WO 00-04192),corresponding to U.S. patent application Ser. No. 09/744,097 filed Jan.16, 2001 entitled “Methods for Detecting and Mapping Genes, Mutationsand Variant Polynucleotide Sequences,” which is hereby incorporated byreference herein for all purposes, methods and compositions for modularprobe and reporter systems that improve the specific detection of genesand mutations and that amplify signaling were disclosed. These disclosedcompositions and methods include:

-   -   1. Probe methods, known as WRAP-PROBEs, that are manufactured        from synthetic DNAs, from PCR (polymerase chain reaction)        products, or from cloning products, wherein the probes have a        central, target-specific sequence that is helically wrapped        around the target strand, and wherein they have one or more        generic linkers at one or both ends that bind one or more        reporters. By binding separate reporters to the ends of the        probes after coiling the probes around the target, the reporters        are more effectively tethered, and they thereby provide far more        effective signaling than is achieved with simple labeled probes.        Indeed, this method can provide multi-fold signal amplification        if dual chains or arrays of long labeled reporters are bound to        a short WRAP-PROBE of this configuration. This WRAP-PROBE        composition also provides an economic advantage in being able to        use generic linkers to interchangeably bind either different        reporters to the same probe or different probes to the same        reporter, wherein a series of generic reporters may be applied        that vary in both the type of signaling and in signaling        intensity.    -   2. Generic reporter methods and compositions such as GENE-TAGs        and TINKER-TAGs, these reporters include liner segments of        double stranded DNA or chained and joined polynucleotides with        single stranded linkers at one or both ends that can join        together in arrays and can join to the linkers of WRAP-PROBEs or        related probes to provide amplified signaling.    -   3. DNA-based connectors called Multi-LINKERs, including singular        or composite polynucleotide structures that join to the linker        of a probe and provide two or more secondary linkers in order to        bind multiple reporters to a probe.

The related WRAP-PROBE methods and compositions are suitable for makingtargeted probes that amplify signaling and that more efficiently map ordetect a specific gene sequence in a variety of detection formats suchas in situ gene mapping, dot blots, etc. In those formats, the target ortargets are on the substrate and a small number of labeled probes areindividually manufactured in excess quantity to find and label thosespecific targets. The object is simply to put label on the target,thereby mapping or counting the targets. However, those methods are notsuited for DNA chip or microarray gene expression formats where the chipsubstrate is in fact a set of capture probes and where the probesapplied to the chip are the true targets of the assay. Thus the objectof an expression assay is to determine the relative frequency of themRNA transcripts in the original tissue sample, and the array is just adevice to capture and count a labeled probe set derived from the sample.Thus this probe set must maintain its relative frequencies—accuratelyrepresenting the thousands of different gene transcripts in the originaltissue. Consequently, WRAP-PROBEs for expression array analysis cannotbe individually manufactured in the same way as prior WRAP-PROBEs wereseparately tailored to specific genes.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions of matter thatallow quantitative, sensitive and rapid analysis of gene expressionpatterns in different cells and tissues as a means to detect functionalchanges associated with development and physiology, to diagnose abnormalvariations related to disease, and to discover and assess pharmaceuticalagents. The invention is designed for and particularly suited tomultiple analyte formats such as cDNA chips and expression microarrayswhere the diagnostic value would be improved by increased signaling andby determining the true frequency of different mRNA transcripts in asample and not just their approximate frequency—a standard poorlyaddressed by current methods. The invention is complementary to priorinventions of the applicant which provide a probe construction, known asWRAP probes, for detecting genes and nucleotide sequences, which employgeneric reporters such as GeneTAGs or TinkerTAGs that are linked toterminal linkers of the probes, and which may employ multi-linkercomponents to join multiple reporters to each probe.

The present invention employs novel primer, linker, adapter, extenderand reporter compositions and molecular processing methods to globallytransform a mixed pool of mRNAs into a pool of modified cDNA-basedprobes, called WRAP-Probes, that have common universal linkers at one orboth ends for joining reporters, to thereby provide more definedsignaling as well as greater signaling potential. The basic principle ofthese methods is to achieve signaling by affixing generic reporters tothe ends of the probe, either directly or via terminal linkers, ratherthan by labeling the target specific segment which varies in size foreach gene. This invention thus allows quantitative analysis ofexpression since the signaling element is effectively equalized for eachtranscript detected, and it improves sensitivity since reporters can beaffixed that have greater signaling potential than a labeled probe.Alternate embodiments of the probes, the multi-linking units, and thegeneric reporters have been devised and these components can be usedtogether in a modular manner to achieve different detection andsignaling objectives.

When the set of WRAP-Probes is constructed with common universal linkerson both ends, this configuration creates an opportunity to use theselinker sequences as global primers, thereby allowing the duplication ofthe entire pool of probes by exponential amplification procedures suchas PCR. Alternate amplification methods were invented that produceeither singular WRAP-Probes from each mRNA transcript or a fragmentseries of smaller WRAP-Probes from each transcript. These methodsinclude novel compositions and procedures to create truncated probes andto affix double-linker/primer sites so that they can be reliablyamplified by exponential methods. The probes are then globally amplifiedand labeled during PCR with a single primer set. These amplified probescan also achieve signaling quite simply and inexpensively with newcompositions called ChipTAGs that are composed of one or more labeledpolynucleotides which additionally serve both linker and primerfunctions. Thus effective methods were devised that transform a mRNApool into a set of smaller probe subunits which are globally amplifiedand suitable for the analysis of gene expression with cDNA chips ormicroarrays. These methods and compositions improve the quantificationof gene expression and allow highly improved detection of raretranscripts and or very small samples.

To overcome the difficulties of current compositions and methods and tostill obtain the signaling advantages of the WRAP-PROBE invention, inthe present invention, reagents and probe systems that extend theWRAP-PROBE design to expression array applications by globallyconverting a complex pool of mRNA transcripts into a pool of probeshaving common universal linkers on one or both ends have been developed.However, the present invention differs compositionally from the priorinvention because the functional product of this invention is not asolitary WRAP-PROBE, but in fact a composite set of WRAP-PROBEs thatnecessarily contains multiple probes of considerable diversity withimportant differences in relative frequency. While it would be possibleto make individual WRAP-PROBEs from single genes in a pool of mRNAproducts in the same manner as RT-PCR is applied to individually copyand amplify a single mRNA gene product and determine its presence, suchan approach would be costly, inefficient and would introduce bias. (Thisapproach would require the manufacture of gene specific primers for eachgene target wherein the primers would additionally have a universallinker on their 5′ end.) Therefore, the present invention devises anddiscovers a composite probe set of WRAP-PROBEs wherein the probe setshares common universal linkers that enable the joining of commonreporters and that enable the global exponential amplification of theprobe set. Because of the high number of diverse probes involved, it isimportant that the universal linkers applied to the probes do not bindnon-specifically to target sequences on the chip. The present inventionadds an important second function, exponential amplification, to theuniversal linkers of the WRAP-PROBE configuration, and it additionallyprovides methods to globally create and amplify the probe set as acollection of probes. To distinguish this probe set composition, thesenew probes were intentionally called REX-WRAP probes in the applicant'sU.S. Provisional Patent Application Ser. No. 60/187,982, filed Mar. 9,2000, entitled “Methods to Quantify and Amplify Both Signaling andProbes for DNA Chips and Gene Expression Microarrays”, which is herebyincorporate by reference herein for all purposes, to indicate theirdifferent source, form and function as a collection or set ofRNA-derived gene expression probes. However, for linguistic fluidity inthis present description, this probe set devised for expression arrayswill simply be termed WRAP-Probes.

A basic principle of the present WRAP-Probes invention is to achievemore sensitive and quantitative results with expression arrays by addingequivalent reporter signaling to the terminal linkers of the probe set.This approach contrasts with the current practice of labeling the probesinternally—a method causing length-related bias in signaling. This endlabeling approach equalizes signaling per probe and provides a truercount of transcript frequency, and it also allows far greater signalingper probe by adding multiple reporters. Additionally, the dynamic rangeof linear signaling is improved since the standard method can saturatesignaling early for those genes that are both long and abundant.Moreover, other advantages can accrue from not labeling the targetspecific segment with bulky signaling molecules that are poorlyincorporated, such as Cy3 and Cy5. And finally, it is known that thetarget strands of cDNA spotted arrays lie side by side in tight clustersmaking probe hybridization more difficult with large signaling moleculesattached to the bases [Duggan et al., Nature Genetics, 21: 10-14 (1999].Notwithstanding these considerations, labeling can still be applied tothe probes directly to provide additional signaling.

Applicant has devised alternate embodiments of the WRAP-probes method aswell as alternate embodiments wherein probe sets are combined withdifferent reporters or intermediate linkers. To this end, applicant hasdevised and discovered different universal linkers that provide probesets that will bind different reporters, thereby enabling comparativeanalysis of different probe sets from different samples on the sameexpression array.

The most elemental version of the WRAP-Probes method is to create probeswith a single universal linker on one end to enable the binding of ageneric reporter, such as a GeneTAG or TinkerTAG reporter (previouslydescribed as GENE-TAGs and TINKER-TAGs in more detail in InternationalPatent Application Serial No. PCT/US99/16242). Applicant has devised anddiscovered a preferred embodiment of this method by copying the mRNAfrom the 3′ poly-A end by reverse transcriptase (RT) using a modifiedpoly-T primer with universal linker sequences added to the 5′ end. Theterminal linker thus created provides a binding site to attach reporterseither before or after the probe is hybridized to the expression array.The resulting probes are called One-Linker WRAP-Probes. A variety ofsuch modified poly-T primers are devised to allow a multiplicity ofreporter attachments.

Applicant has devised alternate methods that produce a probe set withtwo linkers, known as Double-Linker WRAP-Probes. These methods similarlycreate single-stranded or double-stranded cDNA probe components with amodified poly-T primer having a 5′ universal linker, and then a secondlinker is added to the opposite end so that reporters can bind to twolinkers—pulling on the helically bound probe from both ends as with aprior WRAP-PROBE. This double-linker configuration provides a structuraladvantage for tethering longer or multiple reporters, and itadditionally enables the amplification of the probe set. Applicant hasdevised and discovered several methods and compositions for creatingsuch Double-Linker WRAP-Probes based on joining novel adaptercompositions to the 3′ end or based on applying novel extendercompositions to extend the 3′ end and form a second universal linker.

Several types of adapters and extenders have been devised anddiscovered. Adapters consist of paired polynucleotides joined togetherbut with a single stranded overhang, wherein the overhang provides abinding site to join the adapter to a DNA segment with a complementarycohesive end, and wherein the paired segment provides appended sequencesthat serve a recognition, joining or primer function. The adapters ofthe present invention have universal linker sequences in the pairedsegment, and they differ in the overhang. One type of adapter of thepresent invention, called a Specific Adapter, has a small overhangspecific to a restriction cut site. Another type, called a RandomAdapter, has an overhang of a few random bases. A third type, called aHomopolymeric Adapter, has an overhang of poly-C or poly-G sequences.These adapters are designed to join and ligate onto the 3′ end of a cutor modified probe segment to form a second universal linker. Theextenders of the present invention consist of a polynucleotide withuniversal linker sequences on their 5′ end and a 3′ end with eitherrandom or homopolymeric sequences. The homopolymeric extender of thepresent invention has 3′ poly-C or poly-G sequences and is joined to a3′ probe end of complementary poly-G or poly-C sequences formed withterminal transferase, whereupon the 3′ end of the probe may be furtherextended with the universal linker sequences using the extender productas a template. Alternatively, the present invention provides a novelextender with a random 3′ end that is used in a similar manner exceptthat it can join anywhere along the probe. It only functions as anextender in the present invention when it joins to the 3′ end of theprobe via the random sequences, whereupon the universal linker sequenceprovides a template for polymerizing a 3′ extension of the probe toprovide a second linker end. In the present invention this specialextender, called a Random End-Linker, is employed with a novel procedureof the invention, called Back-Tagging, whereupon repeated thermalcycling steps similar to PCR are employed to make many attempts atputting the Random End-Linker at the far 3′ end of the probe to extendit, wherein the Random End-Linker is preferentially modified at the 3′end to block forward polymerization on the probe template. Consequently,the Random End-Linker preferentially back-extends the 3′ end of theprobe to form a second universal linker and it avoids making partialcopies of the probe itself by forward polymerization.

The above adapter and extender compositions and related procedures ofthe present invention enable the simultaneous global application of asecond universal linker to the 3′ ends of the probe set to formDouble-Linker WRAP-Probes. While such Double-Linker probes can bind atleast twice as many reporters as One-Linker probes, either version givesequivalent signaling per transcript within a sample, and thus truecounting of gene expression frequencies. Where true transcript countingmay be sacrificed for greater sensitivity, applicant has also devisedsecondary embodiments of the Double-Linker WRAP-Probe methods describedabove, wherein multiple short probes are created from each mRNAtranscript, either by fragmenting the RT products or cutting them withrestriction enzymes, and by employing various adapters or extenders toconstruct a series of short WRAP-Probes from them. Applicant has alsodevised alternate embodiments of these probe variants wherein thelinkers are pre-attached to labeling agents, multi-linkers, or reporterconstructs.

Applicant has also devised variations of these fragmented probeprocedures to apply to the original WRAP-PROBEs method for detectingsingle genes or sequences in several in situ hybridization formats suchas RNA arrays, single tissue preps or tissue arrays, [Kononen et al.,Nature Medicine 4: 844-47 (1998)] as well as for the mapping ofparticular gene sequences in genomic DNAs, nuclei and chromosomes; e.g.FISH mapping (fluorescent in situ hybridization). In such cases, clonedor PCR copies of specific genomic DNA or mRNA targets are transformedinto a subset of mini-WRAP-Probes with linkers at one or both ends.Applicant has devised several embodiments by cutting the full lengthprobe components into smaller segments with restriction enzymes,shearing, RNase enzymes and the like and then universal GeneTAG linkersare applied to one or both ends by modifications of the above mentionedprocedures for putting the second universal linker on the 3′ end ofDouble-linker WRAP-Probes. The hybridization of these fragment probes totarget tissues provides multiple adjacent probes along a target, andthus highly amplified signaling since each probe can bind one or moregeneric reporters (e.g. GeneTAGs) with greater signaling capacity than asimple labeled probe.

Applicant has also devised WRAP-Probes that are created with multiplelinkers and or multiple reporters pre-attached to one or both ends.These configurations are achieved by attaching generic reporters such asGeneTAGs or TinkerTAGs to a Multi-Linker or by attaching smallersignaling elements directly to the distal linkers of a Multi-Linkerunit.

Applicant has devised and discovered signaling compositions, calledChipTAGs, which are short polynucleotides conjugated to one or morelabeling agents, that serve as a reporter joined to a universal linkerand that additionally serve a primer function. Similar short reportercompositions called OligoTAGs, that only served a linker and labelingfunction, were previously described in International Patent ApplicationSerial No. PCT/US99/16242 as end-labeled oligonucleotides that weresecondarily joined to a Multi-LINKER unit. The advantages of using theseChipTAG components as linkers, primers and reporters are improved costand efficiency. When bulky fluorescent compounds such as Cy3 or Cy5 arejoined to nucleotide reagents for enzymatic incorporation, they areextremely expensive and they are poorly incorporated into probe orreporter units (1-2% efficiency). In contrast, the same or similarlabeling agents can be chemically conjugated to an oligonucleotide orpolynucleotide more reliably (98-99% efficient) and both reagent costand manufacturing are relatively inexpensive.

Applicant has also devised two or more sets of GeneTAG, TinkerTAG,ChipTAG and Multi-Linkers, with different linkers and differentlabeling, so that two or more samples can be labeled differently andsimultaneously compared on an array to determine relative differences inexpression levels between samples.

Applicant has also devised modified poly-T primers to generateWRAP-Probes that are pre-attached to one or more direct or indirectsignaling elements, that are pre-attached to Multi-LINKERs, with orwithout signaling elements attached, and/or that are pre-attached tolabeled GeneTAGs, TinkerTAGs or other generic reporters. The mostelemental of these dual function compositions are a modified poly-Tprimer with a label agent such as Cy3 or Cy5 conjugated to the 5′ end ofthe primer, with a preferred embodiment having a universal linkersequence on the 5′ labeled end to add further reporters. The advantageof these methods is that by joining probes and reporters beforehand, oneor more hybridization step can be eliminated.

Applicant has also devised WRAP-Probes that employ either modifiedpoly-T primers, Multi-LINKERs, GeneTAGs, TinkerTAGs or ChipTAGs, thatare not based on fluorescent or radioactive labeling, but rather, theyare labeled with refractory or light scattering particles or withmetallic or semiconductor based signaling elements—alternativelyallowing the detection of microarrays or DNA chips with novel optical orphotonic sensors or with micro-electronic circuits or sensors.

The above-described Double-Linker WRAP-Probe methods also allow a majormethodological departure from the general principle of creating labeledcDNA probes from each mRNA transcript. Namely, when a universal linkersequence is created on both ends of each probe, those sequences can bedesigned and used as generic primer sites for globally copying andamplifying the entire pool of probes with a single primer set or evenwith a single primer using common PCR methods or related processes.Applicant has devised and discovered methods to make such globallyamplified WRAP-Probe probe sets. These methods employ in part one of theabove described double-linker compositions and procedures (based on theligation of adapters or the annealing of extender templates) to apply asecond linker to the first strand cDNA copy which already has a firstlinker created by the modified RT primer. However, these copying methodsare modified for global amplification since exponential PCR of fulllength copies, particularly of the longer transcripts, may produce biasand deficiencies in the amplification products as described above.Therefore, two preferred WRAP-Probe amplification procedures have beendevised; 1) to make a single WRAP-Probe from a shortened 3′ end of eachtranscript, or 2) to cut and transform full length or near full lengthcDNA copies into a set of multiple short probes called Mini-WRAP-Probes.Either of these procedures produce a pool of short probes all havinggeneric linker/primer sequences at each end so that they are suitablefor exponential amplification.

Therefore, the basic principle of the amplified WRAP-Probe method is toconstruct or reconstruct RT generated cDNA probes as short or shortenedprobes of similar length, with generic linkers on both ends that provideuniversal primer binding sites independent of gene specific sequences,so that the entire set of mRNA derived probes can be globally amplifiedby PCR in a unbiased manner. This invention provides several importantadvantages. First, expression analyses can be conducted on very smallRNA or tissue samples. Second, quantitative signaling can be preservedeither by attaching generic reporters to the ends of the amplifiedprobes or by shortening the probes to approximately the same length sothat internal labeling becomes more equalized between genes. Third,since all products are amplified with a single primer set, all templateshave essentially equalized access to primers, and thus any bias towardsamplification of the more abundant transcripts is reduced or eliminated.Fourth, by amplifying the probes, more limited or economic signalingmethods can be employed such as ChipTAGs since the number of labeledprobes can be exponentially increased. And finally, the amplified probeset effectively increases the concentration of the sample thus allowinglarger chip formats—that is the same tissue or RNA sample can producesufficient probes to cover a larger chip hybridization area enablingsimpler, less miniaturized, and less expensive chip manufacturingprocesses.

Applicant has devised and discovered three primary new methods toglobally achieve short double-linker probes from mRNA that are suitablefor PCR amplification: 1) restriction cutting and adapter ligation, 2)globally truncated RT and probe extension with a random end-linker, and3) globally truncated RT and random adapter binding. These methods arebased upon and modified from the Double-Linker WRAP-Probe methodsdescribed above.

For the first sub-method, applicant has devised and discoveredprocedures to achieve short probes by cutting the initial cDNA productswith one or more restriction enzymes; capturing the cut fragments fromthe poly-A end; and ligating a matching adapter to the opposite end toprovide the second linker/primer sequence needed for PCR amplification.Since the cut sites vary for different genes, enzyme selection issignificant, and thus it is preferred that two enzymes are employed inseparate samples to ensure that no gene lacks representation in theprobes.

For the other sub-methods, applicant has devised and discoveredprocedures to globally achieve short probes of similar length bydramatically truncating the RT protocol, from the typical exposure timeof one or two hours, down to brief exposures of a few minutes or less.Standard RT protocols, including manufacture of probes for DNA chips,are typically based on one or two hour RT exposures to ensure that thefull length of all transcripts is copied since prior work hadestablished that 95% of RT copying is completed in about 50 minutes[Verma et al., Nature New Biology, 235: 163-169 (1972); Verma et al.,Biochem. Biophys. Acta, 473: 1-38 (1977); Gubler et al., Gene, 25: 263(1983)]. However, by radically cutting the RT enzyme extension time downto a period of minutes or seconds, applicant has discovered that the RTproducts are truncated prematurely in relatively equivalentlengths—producing a pool of cDNA probes that are randomly andarbitrarily short—regardless of the gene length of the original mRNAtranscripts. This novel protocol, called Short-RT, in effect equalizesthe length of all probes to a narrow size range dictated somewhatrandomly by when each transcript starts the copying process relative tothe instant that RT enzyme exposure begins. The resulting pool ofrandomly short products, that are mostly hundreds of bases long vs.thousands of bases long, are easily amplified by PCR. More importantly,the known bias that occurs in amplifying different gene products ofdifferent length is effectively overcome by this random length samplingmethod. This key modification, Short-RT, provides a simple, economicmethod to remove an important barrier to unbiased exponentialamplification, gene length variation, which has inhibited the priordevelopment and use of PCR-based protocols for DNA chip applications aswell as other global mRNA comparisons.

Therefore, for the second sub-method, globally truncated, Short-RTproducts are prepared with a first universal linker, and then theextender reagents described above as Random End-Linkers are applied tocreate a second universal linker. While these extenders can bindanywhere along a probe, a significant result only occurs when they bindat the 3′ end, wherein that 3′ probe end is back extended with a linkersequence that forms a primer binding site. Therefore, the protocolcalled Back-Tagging described above was devised and discovered toincrease the opportunity for such an end extension to occur. This novelprotocol commonly employs rapid thermal cycling for approximately 100 to200 cycles that mimic the steps of PCR (1. denaturing at hightemperature, 2. annealing at low temperature, and 3. briefly extendingat moderate temperature) but do not practice PCR since the 3′ end of theRandom End-Linker is typically blocked. Whereupon, the extenders arerepeatedly hybridized to the probe to extend it with a universal linker,thereby providing the second linker/primer site needed for subsequentPCR amplification of the probes. Moreover, since the probes are ofrandomly truncated length, they can also be internally labeled, duringor subsequent to PCR amplification, instead of or in addition to endlabeled, without reintroducing signaling bias between different genes.

For the third sub-method, globally truncated, Short-RT products areagain prepared with a first universal linker, and the second linker isthen applied to the 3′ end of the probes with the novel Random Adapterdescribed above which has a short random overhang. The random segmentprovides a random binding mechanism to anchor the adapter on the 3′ endof any probe so that probe and adapter components can be ligatedtogether. After ligation, the adapter-probe complex is denatured andpurified to release the unbound half of the adapter—thus providingprobes containing linker/primer sites on both ends suitable for PCRamplification. For the same reasons as above, this sub-method alsoallows the probes to be internally labeled, instead of or in addition toend labeled, without reintroducing signaling bias between differentgenes.

Elements of the above sub-methods can also be combined together indifferent ways or combined with pre-existing technologies toalternatively produce double-linker is probes from single or doublestranded cDNA probes made with a 5′ first linker. For example, globallytruncated, Short-RT products can be prepared with a modified RT primer,converted to double stranded cDNA probes, and joined to commercialadapter/linker products, e.g. Clontech's Smart or Marathon adapterproducts, to create a 3′ second linker site on the probes.Alternatively, globally truncated Short-RT products can be prepared witha first universal linker using the Modified Poly-T primer, and then, ahomopolymeric 3′ tail of poly-C or poly-G sequences is provided [Ivanovaet. al., Nucleic Acids Res. 23: 2954 (1995)]. Thereafter, an extenderpolynucleotide with a 3′ matching homopolymeric segment is applied tocreate a second universal linker. Alternatively, an adapter could bemade with a 5′ universal linker and a homopolymeric 3′ end that wouldallow this product to be ligated to the 3′ end of the probe creating asecond universal linker. These alternate procedures provide additionalmethods to affix the second linker/primer sequence required for globalPCR amplification of the probe set.

Finally, the applicant has devised adapters and extenders applicable tothe above methods that provide second linkers on the 3′ end that mirrorthe first linker sequences of the 5′ end whereupon the probes can belinked similarly via each end as well as amplified by PCR with a singleprimer rather than a pair of primers.

With these new methods, the primers used for global amplification of theWRAP-Probe probe set or the Mini WRAP-Probe probe set can employ simpleChipTAG compositions to generate probes with pre-attached terminallabels. Due to exponential amplification of the probes, such limitedsignaling can be quite sufficient. Alternatively, the primers can bepre-attached to multi-linkers or reporters such as GeneTAGs that providegreater signaling per probe. Either approach will allow a singlehybridization step to apply both probes and reporters.

Current art has not been able to employ exponential amplificationmethods for expression arrays because of one or more of thefollowing: 1) the need for multiple unique primers, 2) the variability'sand deficiencies of employing hexamers or random primers as a substitutefor unique primers, and 3) the great variation in gene transcript sizewhich can alter amplification characteristics. The amplified WRAP-Probesand Mini-WRAP-Probes devised and discovered here set a new precedent insignaling potential for expression arrays. By creating one or multipleshort probes from each transcript and by using one universal primer setfor the entire pool of probes, the bias of global amplification isavoided. Moreover, the creation of globally short probes providesconsiderable advantages for application to DNA chips or microarrayssince short probes improve both the kinetics of hybridization and accessto small target opportunities on the chips. The WRAP-Probe methodproduces one amplified probe product per transcript and thus preservesthe principle of generating equivalent signaling per transcript as withnon-amplified WRAP-Probes. The Mini-WRAP-Probe method relaxes thatprinciple for the sake of simplicity and greater signaling, and yet itstill does not depart further from the signaling differences pertranscript that are inherent in the current art of labeling probes alongtheir length. This Mini-WRAP-Probe method is also well suited toexpression arrays based on specific oligonucleotides on the chip vs.spotted cDNA, and in that case the multiple products from eachtranscript does not bias signaling since each oligonucleotide segment onthe chip is known and accounted for separately. In any case, these newmethods provide the first global procedures to amplify mRNA derivedprobes for gene expression arrays in an exponential manner.Consequently, these methods are highly advantageous over current artthat requires large amounts of mRNA per each microarray assay. Incontrast, these exponential amplification methods allow the analysis ofvery minute samples that may be available from micro dissections, needlebiopsies, small blood samples, forensic traces or archived tissue aswell as repeated analysis of the same sample. These advantages areparticularly relevant for clinical or forensic specimens where only asingle, small sample may be available. Finally, these global probeamplification procedures will allow the repeated testing of geneexpression changes over time due to development, disease, or inducedresponses to drugs or therapies.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one embodiment of the inventionand together with the description, serve to explain the principles ofthe invention.

FIG. 1: One Linker WRAP-Probe method. FIG. 1 depicts the creation ofcDNA probes with a universal linker from mRNA transcripts and applyingthem to provide amplified and quantified signaling. Step 1 depictsbinding the modified poly-T primer with a universal linker to the poly-Atail of mRNA and polymerizing first strand cDNA probes with a universallinker; Step 2 depicts binding the probes to a cDNA chip; Step 3 depictsbinding labeled GeneTAG reporters to the universal linkers of theprobes.

FIG. 2: Amplified WRAP-Probe method. Sub method One: Restriction cuttingand adapter ligation. Step 1 depicts the conversion of mRNA into doublestranded cDNA with one universal linker by copying the mRNA with RT anda modified poly-T primer, and by polymerizing a second strand with DNApolymerase and RnaseH. Step 2 depicts cutting the probes with arestriction enzyme and capturing them with magnetic beads via thecapture moiety, such as biotin. Step 3 depicts ligating the SpecificAdapter to the cut ends of the probe to provide a second universallinker and to form double-linker probes. Step 4 depicts PCRamplification of the probes, wherein the probes are either labeledinternally with labeled bases or labeled on the end using labeledprimers eg. ChipTAGs. Additionally, GeneTAG or TinkerTAG reporters canbe added to the probes after they are bound to the cDNA chips.

FIG. 3: Amplified WRAP-Probe method. Sub method Two: Applying Short RTand the Random End-Linker to make Double-Linker cDNA probes. Step 1depicts how the mRNA is converted to first strand cDNA probes with oneuniversal linker and it further depicts the short RT procedure wheredifferent transcripts of different lengths are stopped short atapproximately the same length. Step 2 depicts binding of the randomextender, called the Random End-Linker, to the probes during multi-cyclethermal cycling where the extender binds but does not prime if it bindsanywhere along the probes except the 3′ end, and where it extends the 3′end with a universal linker when it binds to the 3′ end. Step 3 depictsthe further step of amplifying the double linker probes by PCR withlabeling incorporated either in the bases or by using ChipTAG primers.Labeling can be applied in both ways to the probes, and additionallabeling can also be provided by adding GeneTAGs or TinkerTAGs to theuniversal linkers of the probes.

FIG. 4: Amplified WRAP-Probe method. Sub method Three: Applying Short RTand Random Adapter to create double-linker probes. Step 1 depicts howthe mRNA is converted to first strand cDNA probes with a 5′ universallinker by polymerizing cDNA with a modified poly-T primer, and itfurther depicts the short RT procedure where different transcripts ofdifferent lengths are stopped short at approximately the same length.Step 2 depicts binding of the random adapter by ligation to the 3′ endof the probes to form double linker probes. Step 3 depicts amplifyingthese double linker probes by PCR with labeling incorporated either inthe bases or on the ends by using ChipTAG primers.

FIG. 5: Amplified WRAP-Probe method. Sub method Four: Short RT andhomopolymeric. These methods use either homopolymeric adapters orextenders. Step 1 depicts how the mRNA is converted to first strand cDNAprobes with one universal linker, and it additionally depicts the shortRT procedure where the first strand copy is stopped short for alltranscripts. Step 2 depicts alternatively either the binding of thehomopolymeric extender on the left or the homopolymeric adapter on theright to extend or append the second universal linker unto the 3′ end ofthe probes to form double linker probes. Step 3 depicts the further stepof amplifying these double linker probes by PCR with labelingincorporated either in the bases or on the ends by using ChipTAGprimers.

FIGS. 6A-6B: Images from Example 2. FIG. 6A depicts the probeshybridized to the chip that are internally labeled with Cy3 (green).FIG. 6B, the lower portion, depicts the GeneTAGs hybridized to theprobes of 6A above wherein the GeneTAGs are labeled with Cy5 (red) andshowing increased signaling with GeneTAGs. In this document, the colorarray images were converted to black and white and inverted since thearray images are artificial, scanned pseudocolor images not truephotographic images.

FIG. 7: Image from Example 3. FIG. 7 depicts PCR amplified probeshybridized to the chip that are internally labeled with Cy3 (green).This image is also converted to black and white and inverted from apseudocolor green image.

FIG. 8: Image from Example 3. FIG. 8 depicts GeneTAGs hybridized to theprobes of FIG. 7. wherein two layers of GeneTAGs are applied and theGeneTAGs are labeled with Cy5 (red) showing increased signaling. Thisimage is also converted to black and white and inverted from apseudocolor red image.

FIG. 9: Image from Example 4. FIG. 9 depicts a small sample of theprobes from Example 2 above that were re-amplified by PCR and applied toanother expression microarray. In this case a “Red” ChipTAG primer wasemployed as a single primer to globally amplify and label all the probeproducts. Thus labeling was achieved from a single Cy5 fluor that isconjugated to the 5′ end of the ChipTAG primer. Additional ChipTAGprimer was added back to the probe sample after PCR amplification toincrease signaling. This image is also converted to black and white andinverted from the red pseudocolor image.

FIG. 10: Images from Example 6. These images demonstrate the use ofAmplified WRAP-Probe Sub method Two which employs the Short RT andRandom End-Linker procedures followed by PCR amplification of theprobes. In this case, gene expression analysis is shown with P-32labeled probes that are hybridized to membrane-based arrays. Expressionprofiling is demonstrated that distinguishes between control and IL-13treated monocyte samples based upon starting with only 1 microgram oftotal RNA per sample.

FIG. 11: Images from Example 7. These images shows P-32 labeled probeshybridized to membrane-based arrays using the Amplified WRAP-Probe Submethod Three that employs the Short RT procedure, the ligation of RandomAdapters, and then PCR amplification of the probes. Expression profilingis depicted for Short RT using different RT exposure times of 2 min, 5min, 10 min and 20 min. The shorter RT exposure times give betterresults than longer exposures.

DETAILED DESCRIPTION OF THE INVENTION

Preferred embodiments of the invention are now described in detail.Referring to the drawings, like numbers indicate like parts throughoutthe views. As used in the description herein and throughout the claimsthat follow, the meaning of “a,” “an,” and “the” includes pluralreference unless the context clearly dictates otherwise. Also, as usedin the description herein and throughout the claims that follow, themeaning of “in” includes “in” and “on” unless the context clearlydictates otherwise. In the foregoing discussion, the following termswill have the same meaning as provided in International PatentApplication Serial No. PCT/US99/16242 except as modified and/or expandedherein unless the context clearly dictates otherwise.

-   -   WRAP-Probe: a single DNA based probe affixed with universal        linkers on one or both ends to bind generic reporters.    -   WRAP-Probe probe set: a pool of WRAP-Probes made from a pool of        mRNA species to represent and detect relative RNA transcript        frequencies with gene expression arrays.    -   One-Linker probes: WRAP-Probes with one universal linker.    -   Double-Linker probes: WRAP-Probes with universal linkers on both        ends.    -   Amplified WRAP-Probes: a pool of WRAP-Probes exponentially        amplified by PCR or related processes.    -   Mini-WRAP-Probes: a series of small WRAP-Probes made from        fragmenting first strand cDNAs from a pool of mRNAs.    -   GeneTAG: linear generic reporter molecules with terminal        universal linkers.    -   TinkerTAG: GeneTAGs constructed of partially overlapping        polynucleotides that self assemble, with or without single        stranded arms for binding labeled oligonucleotides.    -   ChipTAG: small multi-function labeled universal linker that also        serves as a primer.    -   Universal Linker: a single stranded nucleotide sequence that        allows the joining of two probe and or reporter elements by        complementary nucleotides while the linker sequences are not        complementary to the target sequence.    -   Multi-Linker: a polynucleotide or complex of polynucleotides        that self assemble and that provide a probe linker and two or        more reporter linkers.    -   Modified Poly-T Primer: a global poly-T primer for RT reactions        modified on the 5′ end typically with a universal linker and/or        a capture moiety, label, reporter or multi-linker.    -   Adapter: paired polynucleotides with blunt or cohesive ends for        joining to DNA fragments and providing added functions such as a        linker, primer or reporter binding functions.    -   Specific Adapter: a composite of paired polynucleotides with an        overhang of specific sequences that can be joined to restriction        cut ends of DNA fragments and that provide a universal linker.    -   Random Adapter: paired polynucleotides with an overhang of        random sequences that can be joined to any DNA fragment and that        provide universal linker sequences.    -   Homopolymeric Adapter: paired polynucleotides with a Poly-C or        Poly-G overhang that can be joined to a Poly-G or Poly-C        sequence and that provide universal linker sequences.    -   Homopolymeric Extender: extender polynucleotide with a 5′        universal linker end and a 3′ Poly-C or Poly-G end that can join        to a 3′ Poly-G or Poly-C end of a probe and serve as a template        to extend the probe with a universal linker sequence.    -   Random End-Linker: extender polynucleotide with a universal        linker region on the 5′ end and a random sequence region on the        3′ end that can join to the 3′ end of a DNA segment and serve as        a template to extend that DNA segment with a universal linker        sequence, said extender being preferably modified on the 3′ end        to block capacity for polymerase extension.    -   Probe Modifier: a category representing any of the above        adapters and extenders that apply a universal linker to the 5′        or 3′ end of a probe, including the random adapter and extender,        the homopolymeric adapter and extender and the modified poly-T        primer, as well as including the ChipTAG labeled primers which        add label directly onto to the end of probe when used to amplify        a double linker probe.    -   Short-RT: modified RT protocol in which all products are stopped        short during RT extension to produce similarly short cDNA probes        suitable for PCR amplification.    -   Back-Tagging: modified thermal cycling protocol for applying        Random End-Linkers to back-extend probes using multiple thermal        cycling steps with short extension times.    -   mRNA: messenger RNA transcripts which are a subset of total RNA.    -   cDNA: DNA copies of mRNA    -   Microarray: a miniaturized grid of nucleic acid targets to        detect a pool of probes.    -   cDNA chip: a cDNA based microarray.    -   Expression Array: a grid of nucleic acid targets based on cDNA        or cDNA sequences    -   PCR: polymerase chain reaction to amplify DNA exponentially.    -   RT: reverse transcriptase enzyme method to copy RNA.    -   RT-PCR: reverse transcriptase plus PCR to copy and amplify a        specific mRNA transcript.    -   Hybridize: formation of specific hydrogen bonding interactions        between complementary strands of nucleic acids.    -   Cross-link: covalent linkage between hybridized nucleic acid        strands.    -   PUVA: psoralen plus UVA crosslinking procedure.    -   TA site: nucleotide sequence reading 5′-3′: thymidine, adenine.    -   C9: a spacer that is 9 carbon atoms long.    -   C18: a spacer that is 18 carbon atoms long.    -   UNG: Uracil-Nucleotide-Glycosylase procedure where Uracil bases        are incorporated into DNA to make them labile to glycosylase        digestion.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

The present invention relates to a number of probe compositions,manufacturing compositions, and signaling compositions and associatedmethods that improve the preparation, application and detection ofprobes and reporters for gene expression arrays and relatedmulti-analyte hybridization assays, including but not limited to cDNAchips, oligonucleotide chips, biochips and other microarray formats. Theresent invention is in part based upon or incorporates prior inventionsof the applicant, described in International Patent Application SerialNo. PCT/US99/16242 (WIPO Publication WO 00-04192), the disclosure ofwhich is hereby incorporated by reference in its entirety.

Composition of Matter:

The present invention relates to a universal linker composition suitablefor gene expression arrays and related hybridization assays, including anucleotide linking sequence which can be globally appended to the endsof a set of probes derived from mRNA transcripts of an analyte sample toproduce a probe set where the probes have a common linker at one or bothends. These universal linker sequences are not complementary to thetarget sequences of the assay, and they provide binding sites to jointhe members of the probe set to common reporters. The universal linkersare also suitable for chemical cross-linking between bound linkers, sothat probes, reporters, and any intermediate linking elements, can bepre-attached together and covalently bonded. The universal linkersadditionally serve as universal primer binding sites for copying oramplifying the probe set.

The universal linkers of the present invention are suited to binding avariety of reporters that may have complementary linkers, particularlyreporters such as the GeneTAG and TinkerTAG reporters, and arraysthereof, as referenced and described previously, where the GeneTAGreporters include linear labeled segments of duplex DNA that terminatein single stranded universal linkers and the TinkerTAG reporters thatcontain a structurally similar linear complex of labeled polynucleotidesand that also terminate in single stranded universal linkers. Thesereporters can also form arrays of reporters joined end to end. Theuniversal linkers are additionally suited to binding multi-linkerelements, as referenced and described previously, may include one ormore joined polynucleotides that form a probe linker at one end and twoor more reporter linkers at the opposite end. The probes of the presentinvention may employ the universal linkers to bind reporters directly orindirectly, by virtue of binding multi-linkers to the probes, andbinding reporters such as GeneTAGs or TinkerTAGs to the linkers of themulti-linkers.

The present invention additionally relates to a set of two or moreuniversal linkers containing linker sequences which can bind two or moresets of probes to two or more different common reporters, eitherdirectly or via intermediate linkers, to provide different labeling todifferent sets of probes. These universal linker compositions includebut are not limited to:

a. A first linker sequence 5′CTACGATACGATAGCGCCTAAGAGTAG (Seq. ID.No. 1) and its complement, known as the Red universal linker;

b. A second linker sequence 5′CCTAGACCTACGACATAGGTACCCTAC (Seq. ID. No.2) and its complement, known as the Green universal linker;

c. A third linker sequence 5′CGTAGAACTAGCACGCTACGTACTAGG (Seq. ID. No.3) and its complement, known as the Blue universal linker;

d. A fourth linker sequence 5′GGCTATCGCTACGTAGACTAGACCTAC (Seq. ID. No.4) and its complement, known as the Orange universal linker.

The present invention relates to a probe set composition, calledWRAP-Probes, for gene expression arrays and related hybridizationassays, to provide common equivalent signaling per probe regardless oflength, as contrasted with signaling bias which results fromincorporating label along the length of each probe. This probe setincludes a pool of modified cDNA probes copied in part from a sample ofmRNA transcripts, but appended with terminal universal linkers, as inthe prior WRAP-PROBE invention referenced and described previously,where each single stranded probe of the probe set contains a centraltarget specific segment copied from a single mRNA transcript, and auniversal linker located on a terminal end of the probe. The universallinkers provide binding sites to join common reporters to each probe,and they also provide primer binding sites to copy and amplify theprobes. In a primary embodiment, to allow exponential amplification ofthe probes, probe sets are also made with universal linker sequences atboth terminal ends. The universal linker sequences at both ends may bedifferent or they may mirror one another, in which case the probe sethas a common primer binding site and may be amplified with a singleprimer.

In another embodiment of the WRAP-Probe probe set, calledMini-WRAP-Probes, the probes are fragmented to provide multiple probesper mRNA transcript. Initially, the probe set of first strand cDNAprobes is fragmented and then universal linkers are applied to one orboth ends of the fragments to create a final probe set of multiple shortprobes having universal linkers. Thus, each transcript becomes a seriesof short or Mini WRAP-Probes with one or two terminal linkers, thatprovide greater signaling in two ways, by amplifying the multiple probefragments, and by binding reporters to the linkers of the multiple probefragments. Such fragmentation may be induced randomly by shearing,sonication, RNase, RNase-H, UNG, single strand cutting enzymes, and liketreatments, or alternatively at specific sequences with restrictionenzymes.

In other embodiments, two or more probe sets of WRAP-Probes orMini-WRAP-Probes are provided, having probe sets that can be compared inthe same assay, where the probes of each set have different universallinkers, and where the linkers provide binding sites for differentmulti-linkers, reporters or labeling that distinguish the probe setsfrom one another.

The present invention relates to a series of modified Poly-T Primercompositions for globally initiating the copying and conversion of mRNAtranscripts into a set of WRAP-Probes. In the primary embodiment, themodified Poly-T Primer composition contains a polynucleotide in whichthe 3′ end provides a poly-T primer segment to initiate RTpolymerization and the 5′ end provides a universal linker, wherein thelinker can bind reporters to the probe. Alternatively, the Poly-T Primercomposition has a poly-T end that also contains an anchor sequence topreferentially bind to the forward end of the poly-A segment of mRNAtranscripts. In a primary embodiment, the anchor sequence takes the form5′-poly-T, V, N-3′, where poly-T is a series of thymidine bases, V is avariable base of adenine, cytosine or guanine, but not thymidine, and Nis randomly any base. Other anchor sequences can be employed includingthe sequence 5′-poly-T, V. The Poly-T Primers are preferably made withabout 12 to 20 thymidines in the poly-T segment.

In an additional embodiment, a Poly-T Primer composition is manufacturedwith a capture moiety such as biotin on the 5′ end so that the probeunits can be captured with magnetic beads or other methods and retained,purified, treated, or re-used to copy the original probe set. In otherembodiments, the modified Poly-T Primer is manufactured with labelingelements attached, or alternatively, one or more reporters arepre-attached prior to use. Alternatively, the modified Poly-T Primer isconstructed with a multi-linker pre-attached, wherein reporters can beattached or pre-attached to the multi-linker. Such reporters can includeGeneTAGs, TinkerTAGs or arrays of such reporters. Additionally, a set oftwo or more Poly-T Primer compositions are provided, that includedifferent universal linkers, multi-linkers, reporters and label orlabeling precursor so that each resulting probe subset can bedistinguished by different signaling.

The present invention also relates to a series of adapter compositionsfor providing a second universal linker to the probe sets. One productembodiment is a sequence specific adapter composition, called a SpecificAdapter that is typically ligated to the 3′ end of a DNA probe segment.This adapter product contains two polynucleotides joined together bycomplementary bases, where the complementary bases are a set ofuniversal linker sequences, and where one end contains an additionalsingle-stranded overhang, typically of 1 to about 6 bases, that canspecifically bind to the terminal end of a probe that has been cut witha specific restriction enzyme. These Specific Adapters are alsomanufactured as a set of two or more such adapter products to allowsample comparisons, where each adapter in the set has a differentuniversal linker sequence that can bind different reporters ormulti-linkers.

The present invention also relates to a series of random adaptercompositions for providing a second universal linker to the probe sets.One embodiment is a Random Adapter product that is typically ligated tothe end of a DNA probe segment. The Random Adapter composition containstwo polynucleotides joined together by complementary bases, where thecomplementary bases are a set of universal linker sequences, and whereone end has an additional single-stranded overhang of random bases,typically of 1 to about 6 random bases. Such random sequences, which arealso called degenerate sequences, are typically represented as an “N” insequence descriptions and are chemically synthesized by providingalternatively and randomly: an adenine (A), thymidine (T), cytosine (C)or guanine (G), at each position in the random sequence. A set of two ormore Random Adapters are also provided by the invention to allow samplecomparisons, where each adapter in the set has a different universallinker sequence. Another product embodiment is a homopolymeric adaptercomposition that is also typically ligated to the end of a DNA probesegment, but in this case, the 3′ end of the probes are first extendedwith a poly-C or poly-G sequence. The homopolymeric adapter productcontains two polynucleotides joined together by complementary bases,where the complementary bases are a set of universal linker sequences,and where one end contains an additional single-stranded overhang ofpoly-C or poly-G bases. The homopolymeric adapter binds to acomplementary tail of poly-G or poly-C sequences that is previouslyappended to the probes using terminal transferase and a sole nucleotide.A set of two or more homopolymeric adapters is also provided to allowsample comparisons, where each adapter in the set has a differentuniversal linker sequence. In alternate product embodiments, theSpecific Adapter, the Random Adapter or the Homopolymeric Adapter islabeled.

The present invention also relates to a series of extender products forproviding a second universal linker to the probe sets. One randomextender composition, called a Random End-Linker, binds to the end of aprobe at random and extends its sequences as a copy of the linkersequences of the extender. This extender includes a single-strandedpolynucleotide with a 5′ end containing universal linker sequences, anda 3′ end containing random sequences, preferably about 4 to about 10random sequences (also called degenerate sequences). In a preferredembodiment, the Random End-Linker is chemically modified on the 3′ endto block or prevent polymerase extension of that end, where onemodification practiced is to add a carbon spacer to the 3′ end.Consequently, this product will not forward copy. The present inventionprovides a set of two or more Random End-Linker products to allow samplecomparisons, where each composition in the set has a different universallinker sequence. An alternate extender product of the present inventionis a homopolymeric extender that includes a single-strandedpolynucleotide with a 5′ end containing universal linker sequences, anda 3′ end containing poly-C or poly-G sequences, preferably of about 5 toabout 15 poly-C or poly-G sequences. The homopolymeric extender binds toa tail of poly-G or poly-C sequences that is previously added to the 3′end of a probe by terminal transferase. The present invention provides aset of two or more homopolymeric extender products to allow samplecomparisons, where each composition in the set has a different universallinker sequence.

The present invention relates to a universal linker-primer-reportercomposition, called a ChipTAG, which includes a single-strandedpolynucleotide with universal linker sequences that is manufactured witha label or labeling precursors attached and where the linker sequencesprovide both a primer function for DNA polymerase activity and a linkerfunction to bind the labeled ChipTAG as a reporter to a probe.Additionally, two or more sets of ChipTAG compositions are provided toallow sample comparisons, where the ChipTAGs may differ from one anotherin both their linker sequences and in their pre-attached label orlabeling precursors, and where different labeling is provided todifferent probe sets.

Method:

The present invention relates to a series of methods for gene expressionarrays and related assays that enable the manufacture and application ofthe related composition of matter inventions described above. Thesemethods attach common reporters to the ends of a probe set, typically byvirtue of universal linkers created at one or both ends of the probes,to give each probe in the set an essentially equivalent signaling level,thereby enabling a more effective count of the number of differenttranscripts in the original RNA sample. However, these methods alsoallow internal labeling of the probes by standard methods, eitheradditionally or alternatively. Since some of the methods truncate thelengths of the probes so that their size variation is reduced oreliminated, these methods can additional enable the normalization ofsignaling between probes even when they are internally labeled. Thesemethods can additionally amplify the probe sets globally by virtue ofthe terminal universal linkers so that exponential amplificationprocedures such as PCR or related methods can be practiced if the probeshave linkers at both ends, and so that linear amplification procedurescan be practiced if the probes have one linker.

The present invention relates to a general method to make and applyWRAP-Probe probe sets for gene expression analysis, where more accuratequantitative detection is achieved by attaching common reporters to oneor both ends of each probe, this method comprising:

a. Providing RNA from a tissue sample;

b. Making cDNA probes from the RNA transcripts with universal linkers atone or both ends;

c. Hybridizing the cDNA probes to an array or series of gene specifictargets;

d. Joining reporters to the cDNA probes; and

e. Detecting reporters to determine the expression of genes in thetissue sample.

In its most elementary embodiment, the WRAP-Probe method produces aprobe set with a single linker or reporter end, comprising:

a. Hybridizing a modified poly-T primer with a universal linker to themRNA transcripts;

b. Polymerizing full or partial first strand cDNA copies of thetranscripts to form one linker probes with a common 5′ signaling end.

This method is illustrated in FIG. 1 where step 1 shows the use of thepoly-T primer to make a first strand cDNA probe with a 5′ universallinker, step 2 shows the binding of the probes to the cDNA chip, andstep 3 shows the binding of GeneTAGs to the linkers of the probes.

This one-linker WRAP-Probe method can bind multi-linkers and orreporters to the 5′ universal linker affixed to the probes, includingbut not limited to GeneTAGs, TinkerTAGs or reporter arrays thereof, aswell as ChipTAGs or commercially available reporters such as the bDNAreporters of Chiron Corp. [Urdea et al. (U.S. Pat. No. 5,124,246)] orthe Dendrimer reporters of Polyprobe, Inc. [Nilsen and Prensky, (U.S.Pat. No. 5,487,973)], if such reporters were re-manufactured withnucleotide linking sequences that corresponded to the universal linkersof the WRAP-Probes of the present invention. Alternatively, poly-Tprimer compositions are provided that have multi-linkers and orreporters and or label pre-attached, where a second step is not requiredto hybridize these signaling elements to the probes after the probes arehybridized to the targets of the expression assay. The present inventionalso provides different one-linker probe sets, based upon differences inlinker sequence, labeling and reporter attachment so that probe setcomparisons can be performed on the same assay.

The most common form of the WRAP-Probe method is double-linker probesand the general method to make and apply double-linker WRAP-Probe probesets comprises:

a. Hybridizing the poly-T primer composition with a universal linker tothe mRNA transcripts;

b. Polymerizing full or partial first strand cDNA copies of thetranscripts to form an initial probe set with a common 5′ firstuniversal linker;

c. Affixing a second universal linker to the 3′ end of the probes tomake a final double-linker probe set.

In the above and subsequent double-linker WRAP-Probe methods, each probestrand has a 5′ and a 3′ universal linker, wherein the 5′ end is alreadysuitable for effective end-to-end binding to the complementary 5′ singlestranded linker end of a typical GeneTAG, TinkerTAG or multi-linker.However, the 3′ end is less suitable for such end to end binding.Therefore, based on procedures of the WRAP-PROBE method of the priorinvention, the 3′ ends of the probe set of the present invention areoptionally modified by applying and cross-linking an additionalpolynucleotide linker that reverses the polarity of the probe end toprovide a 5′ universal linker end. (See Example 3) To achieve this, theuniversal linker sequences are typically designed with one or more 5′TAsequences to enable the application of PUVA cross-linking, wherein freepsoralen plus UV blacklight treatment covalently joins contra-lateralthymidine bases. Alternatively, a subset of GeneTAG reporters can bepre-made with 3′ vs. 5′ end linkers in a similar manner, or TinkerTAGscan be made directly with 3′ end linkers.

The double-linker WRAP-Probe method provides several sub-methods toalternatively apply the second universal linker to the 3′ ends of thefirst strand cDNA probes.

The first primary double-linker sub-method of the WRAP-Probe methodemploys restriction enzyme cutting and ligation of the Specific Adapterproduct to shorten the probes and form universal linkers at both ends,wherein the modifications comprise:

a. Providing the Poly-T Primer composition with a capture moiety, suchas biotin, at the 5′ end;

b. Polymerizing first strand cDNA and then second strand cDNA to formdouble stranded cDNA with a 5′ first strand universal linker and acapture moiety.

c. Cutting the double stranded cDNA products with a restriction enzyme;

d. Selectively capturing the terminal 5′ probe fragments of first strandcDNA by virtue of the capture moiety, using strepavidin-coated magneticbeads or similar capture techniques;

e. Joining a Specific Adapter to the cut 3′ end of the captured probefragments to append a 3′ second universal linker and create a finaldouble-linker WRAP-Probe probe set, wherein the probe set is denaturedand applied to gene expression assays.

This method was used to create the double linker probes of FIGS. 7, 8and 9 although those probes were amplified by PCR as well. FIG. 2illustrates this method in step 1, where the mRNA is converted to doublestranded cDNA with one universal linker by copying the mRNA with RT anda modified poly-T primer, and by then polymerizing a second strand withDNA polymerase and RNase H. Step 2 depicts cutting the probes with arestriction enzyme and capturing them with magnetic beads. Step 3depicts ligating the Specific Adapter to the 3′ cut ends of the capturedprobes to append a second universal linker and to form double-linkerprobes. This figure also depicts a further step in Step 4 that is not apart of the above method. In Step 4 the probes are amplified by PCRamplification and either labeled internally with labeled bases orlabeled on their ends using a ChipTAG labeled primer.

In a preferred embodiment of the above method, two or more restrictionenzymes are employed in separate probe aliquots using Specific Adaptersmatched to the cut sites. This modification is provided to ensure thatno gene is unrepresented in a detection sample since the use of onerestriction enzyme may cause a particular gene to always be cut at asite too close to the poly-A end of the transcript to produce a viableprobe. With this modification, the separate probe aliquots are thenmixed and applied together for analysis.

The second primary double-linker sub-method of the WRAP-Probe methodemploys the new random extender product, called a Random End-Linker, toform a second universal linker on the 3′ end of the probes, where theextender is applied with a new thermal cycling procedure calledBack-Tagging. This method comprises the following modifications:

-   -   a. Providing first strand cDNA probes with a 5′ universal        linker;    -   b. Denaturing and removing the RNA;    -   c. Repeatedly hybridizing the random extender to the probes        under rapid thermal cycling conditions similar to PCR, wherein        high temperature DNA polymerase and nucleotides are provided        along with repeat cycles of high temperature denaturing, low        temperature annealing, and moderate temperature but brief        extension, to bind the random extender to the 3′ ends of the        probes via the random segment and to selectively extend the 3′        ends of the probes using the universal linker segment of the        random extender as a sequence template, to create a second        universal linker on the 3′ ends of the probes, to form a final        double-linker probe set.

This method was employed to create the double linker probes of FIG. 10although those probes were also amplified by PCR. FIG. 3 illustratesthis method in step 1 and step 2, where step 1 depicts how the mRNA isconverted to first strand cDNA probes with one universal linker,although this illustration also depicts the short RT procedure where thefirst strand copy is stopped short. Step 2 depicts binding of the randomextender composition, called the Random End-Linker, to the probes duringmulti-cycle thermal cycling where the extender binds but does not primeif it binds anywhere along the probes except the 3′ end, and where itextends the 3′ end with a universal linker when it binds to the 3′ end.Step 3 of FIG. 3 depicts a further step, not a part of this specificmethod, where the double linker probes are amplified by PCR withlabeling incorporated in the bases or by using ChipTAG primers.

Although the Back-Tagging procedure employs thermal cycling and hightemperature polymerase reagents common to the PCR method, it does notpractice the PCR procedure to copy or exponentially amplify the productssince the 3′ end of the Random End-Linker is preferably blocked andcannot serve as a primer. Consequently, when the temperature is loweredeach cycle to anneal the Random End-Linker products to the probes, theseextenders will bind at random but will not function unless they happento bind to the 3′ end of the probe fragment. Indeed, the annealing stepcan be practiced at lower temperatures than would be employed for PCRsince the goal is to force as many Random End-Linkers onto the probestrand as possible to increase the chances that one will bind to the 3′end. Since, the extenders will come off again each high temperaturecycle in an unmodified state they will be reused on the next annealingcycle whereupon they might again bind to the 3′ end. Once they do bindto the end of the probe fragment, it will extend 3′ by polymerizationusing the linker sequences of the Random End-Linker as a template toform a 3′ universal linker. When it binds anywhere else along the probe,the blocked end of the random end-linker prevents copying any portion ofthe first strand fragments. Thus, the formation of spurious fragments isavoided and the rapid use of nucleotides and enzyme is prevented.Therefore, thermal cycling can be continued for hundreds of cycles toeffectively apply second universal linkers to the first strand probes.This principle and method has multiple uses in the present invention andfor other applications.

The third double-linker sub-method of the WRAP-Probe method employs theRandom Adapter product to append the second universal linker, comprisingthe following modifications:

a. Providing first strand cDNA probes with a 5′ universal linker;

b. Denaturing and removing the RNA;

c. Joining the random adapter to the 3′ end of the probes to append asecond universal linker, to create a final double-linker probe set.

This method was used to create the double linker probes of FIG. 11although those probes were amplified by PCR as well. This method isillustrated in step 1 and 2 of FIG. 4 where step 1 depicts how the mRNAis converted to first strand cDNA probes with one universal linker,although this illustration also depicts the short RT procedure where thefirst strand copy is stopped short. Step 2 depicts binding of the randomadapter by ligation to the 3′ end of the probes to form double linkerprobes. Step 3 of FIG. 4 depicts a further step, not a part of thisspecific method, where the double linker probes are amplified by PCRwith labeling incorporated either in the bases or on the ends by usingChipTAG primers.

The fourth double-linker sub method of the WRAP-Probe method employshomopolymeric tailing and application of the homopolymeric adapterproduct to append the second universal linker, comprising the followingmodifications:

a. Providing first strand cDNA probes with a 5′ universal linker;

b. Denaturing and removing the RNA;

c. Extending the 3′ end of the probe fragments with a homopolymeric tailof poly-C or poly-G sequences using terminal transferase and onenucleotide;

d. joining a matching homopolymeric adapter to the homopolymeric tail onthe 3′ ends of the probes to append a second universal linker and tocreate a final double-linker probe set.

Alternatively the homopolymeric extender can be substituted in the aboveprocedure wherein this modification comprises the steps of:

-   -   a. providing first strand cDNA probes with a 5′ universal linker        and a 3′ homopolymeric tail of poly-C or poly-G sequences;    -   b. joining a matching homopolymeric extender to the        homopolymeric tail on the 3′ ends of the probes and polymerizing        a 3′ extension, wherein the universal linker segment of the        extender provides a sequence template for extending the 3′ end        of the probes with a second universal linker sequence, to create        a final double-linker probe set.

These methods using homopolymeric adapters or extenders are illustratedin step 1 and 2 of FIG. 5 where step 1 depicts how the mRNA is convertedto first strand cDNA probes with one universal linker, although thisillustration also depicts the short RT procedure where the first strandcopy is stopped short. Step 2 depicts alternatively either the bindingof the homopolymeric extender on the left or the homopolymeric adapteron the right to extend or append the second universal linker unto the 3′end of the probes to form double linker probes. Step 3 of FIG. 3 depictsthe further step of amplifying these double linker probes by PCR withlabeling incorporated either in the bases or on the ends by usingChipTAG primers.

In preferred embodiments of the WRAP-Probe method, the RT copying of themRNAs is intentionally truncated by greatly reducing the duration ofexposure to the enzyme to purposefully produce very short RT products,generally less than 1000 bases in length and preferably less than 500bases. Brief RT exposure times of several minutes or seconds are hereinemployed as contrasted with one hour or more of RT exposure by standardcDNA chip labeling methods. This radical modification of the RTprotocol, called Short-RT, results in first strand cDNA probe componentsthat are randomly and arbitrarily short such that pre-existing sizedifferences between genes and gene transcripts are effectivelyeliminated, wherein the method normalizes the lengths of the probes,improves the ability to affix random end-linkers to the probecomponents, improves the kinetics of probe binding to the expressionassay, and allows the internal labeling of the probes as a supplement toreporter binding without reintroducing bias in signaling between genesdue to inherent differences in transcript length. Short probes alsoprovide more efficient amplification of the double-linker probes byexponential procedures such as PCR.

The Short RT procedure is generally employed in conjunction with thedouble-linker WRAP-Probe methods described above that don't require aspecific cut site to apply a second linker, wherein the modified stepscomprise:

a. Hybridizing a modified poly-T primer with a universal linker to themRNA transcripts;

b. Polymerizing truncated first strand cDNA copies of the transcripts byabruptly terminating RT polymerase progression by time, to form aninitial set of shortened probe fragments with a 5′ universal linker;

c. Applying a second universal linker to the 3′ ends of the shortenedprobes to create a final double-linker probe set.

In preferred embodiments, the Short RT procedure is improved oraugmented by additional treatments, including but not limited to cold,heat, alkali, enzymes such as RNase and RnaseH, single stranded cuttingenzymes, UNG, shearing, sonication or like treatments.

Amplified WRAP-Probes Methods:

The present invention also relates to a modification of thedouble-linker WRAP-Probes method, called the Amplified WRAP-Probesmethod, to provide improved assay sensitivity, wherein the universallinkers affixed to both ends of the probes are used as primer sites toglobally and exponentially amplify the probe set, the amplificationmethod comprising:

a. Providing a double-linker probe set with universal linker-primersites on both ends;

b. Providing primers that match or complement the universallinker-primer sequences of the probes;

c. Amplifying the set of probes exponentially by PCR or relatedprocesses;

d. Denaturing and hybridizing the probes to an array or series of genespecific targets.

In the Amplified WRAP-Probes methods, probe labeling may be incorporatedenzymatically during PCR either by using bases conjugated directly withlabeling agents, such as Cy3 or Cy5 fluorescent compounds, or byincorporating bases with labeling haptens such as amines, biotin ordigoxygenin, whereupon labeling is added to the haptens in a secondprocessing step. In Examples 2, 3 and 5 below such probe labelingemployed direct labeling with Cy3-dCTP or Cy5-dCTP, or indirect labelingwith amino-allyl dUTP (Sigma) to make amino-conjugated bases, whereuponthe probes are then coupled to Cy dyes using Cy3 or Cy5 mono-functionalreactive dye packs (AP Biotech).

In further embodiments of the Amplified WRAP-Probes method, the probesets are alternatively or additionally labeled via the primers, such asthe linker-primer-reporter products called ChipTAGs that compriselabeled universal linkers. In Example 4 below the ChipTAG primers aremanufactured with Cy5 on their 5′ ends and provide the sole labeling forthe probes. Since ChipTAGs can be labeled more efficiently than probescan be labeled internally, ChipTAG labeling can fully substitute forinternal enzymatic labeling with just a few steps of probeamplification. By labeling the ends of the probes, ChipTAGs also improvequantification of signaling per probe.

Alternatively or additionally, the probes may be labeled by bindingGeneTAG or TinkerTAG reporters to the universal linkers of the probes,generally after the probes are hybridized to the targets on theexpression assay, such as in the Examples 2 and 3 below which employGeneTAGs labeled directly with Cy5-dCTP bases. Additionally,multi-linkers as described previously could be applied to the probes toincrease the number of reporters bound, and furthermore, the labeledprimers, called ChipTAGs can be added to the probes before or after theprobes are hybridized to the targets, so that any matching linker endsnot having label will bind ChipTAGs and add label. This was practiced inExample 4 where an aliquot of ChipTAGs was added back to the probesdenatured and applied to the expression arrays, thereby adding a secondChipTAG to the 3′ end of each single stranded probe.

In a further embodiment of the Amplified WRAP-Probes method, themodified poly-T primer product with a 5′ capture moiety is employed toallow high fidelity re-amplification of the probe set. In thisembodiment, the original double-linker probe products have a 5′ capturemoiety and they are then captured, separated and retained, so that theseoriginal copies may be selectively reused for additional amplificationsof the probe set, such as in Examples 3 and 4 below. This procedure canbe applied to any of the double-linker probe methods to selectivelycapture, retain and reuse the first strand cDNA probes with linkers onboth ends to amplify and re-amplify the first copy of the probes fromthe mRNA transcripts. This procedure inhibits or prevents any bias thatmay be introduced by sequence copying errors during amplification or byany random “Monte Carlo” variations in relative amplification that mayoccur in the very low frequency transcripts.

To improve amplification efficiency and to reduce or eliminateamplification bias, additional embodiments of the Amplified WRAP-Probesmethods are further modified by creating shortened double-linkerWRAP-Probes, generally by employing the restriction cutting or the ShortRT procedures describe above, wherein the probes are generally reducedto less than 1000 bases and preferably to less than 500 bases. Examples3 and 4 below employ restriction cutting to reduce the probe size formore effective amplification. Examples 5, 6 and 7 employ Short RT toreduce probe length, wherein Examples 5 and 6 additionally employ theRandom End-Linker and rapid thermal cycling with the Back-Taggingprocedure to provide the second linker needed for PCR amplification, andwherein Example 7 additionally employs the Random Adapter to provide thesecond universal linker needed for amplification. Run on a gel, theseamplified products form smears of shortened probes in the size rangesdescribed above.

Additional embodiments of the Amplified WRAP-Probes method create andemploy two or more sets of double-linker WRAP-Probes which differ inlabeling, wherein multiple probe sets may be compared in the same assay.

The various WRAP-Probe methods described above, including the single anddouble-linker methods, the amplified methods, and the fragmented probesmethods are additionally or alternatively provided direct signalamplification by applying various reporters to the universal linkers ofthe probe sets, wherein such reporters include, but are not limited to,GeneTAGs, TinkerTAGs, arrays thereof, and multi-linker and reporterconstructs thereof as previously described. In Examples 2 and 3 belowthe probes labeled with Cy3 fluorescence are applied to the expressionarrays and then one or more GeneTAGs labeled with Cy5 fluorescence areadded to each probe in a second hybridization. Therefore, the labelingprovided by the probes and the added labeling provided by the GeneTAGsis evident since the two dyes are separately excited, scanned anddetected in different channels as independent signals.

Other commercial labeling products may also be applied to the universallinkers of the probe sets such as the bDNA products of Chiron Corp., orthe dendrimer products of Polyprobe, Inc. if such products can beprepared with complementary universal linker ends. The present inventionalso embodies the application of commercial labeling agents, such asfluorescent reagents, electron transfer dyes, radioactive isotopes, thecolor emitting quantum dot products of Quantum Dot Corp., gold,Nanogold, or other metallic labeling agents, as well as various labelinghaptens such as amines, thiols, biotin, digoxygenin, dinitrophenol,FITC, etc., wherein such products may be attached to the reagents of thepresent invention including the modified poly-T primers, the variousadapters and extenders and the ChipTAGs, GeneTAGs, TinkerTAGs, andreporter arrays thereof as previously described.

Oligonucleotide based expression arrays, such as the GENECHIPs ofAffymetrix, Inc., have different capabilities and limitations relativeto cDNA based chips. Reflecting these differences, oligonucleotide-basedarrays are less suited for probes made primarily from the poly-A end ofgene transcripts since such oligo-based arrays frequently targetupstream as well as downstream gene regions and fail to score expressionfor a gene if all the oligonucleotides on the chip representing thatgene do not show labeling. Therefore, the present inventionalternatively provides methods to generate probes that better representthe entire gene transcript. The principle of this approach is to copyall or most of the entire transcripts, fragment the cDNA copies to makemultiple probe fragments, and then use the methods and compositionsdeveloped and described here to append universal linkers to all thefragments so that the fragment set can be globally amplified and appliedto expression arrays.

The present invention thus relates to a method for gene expressionanalysis that is devised for oligonucleotide-based arrays, called theMini-WRAP-Probes method, wherein multiple double-linker probes are madefrom each transcript, the method comprising the steps of:

-   -   a. making first strand cDNA probes from a RNA sample;    -   b. fragmenting the probes with a fragmenting agent, the        fragmenting agent selected from the group consisting of        restriction enzymes, RNase, RNase-H, UNG, single stranded        cutting enzymes, shearing, and sonication;    -   c. applying a random probe modifier to the 3′ end of the probe        fragments to append a common universal linker, the random probe        modifier selected from the group consisting of the random        adapter composition and the random extender composition;    -   d. polymerizing a second strand cDNA copy of the fragments with        a primer comprising the universal linker sequence; and    -   e. applying the random extender comprising the same universal        linker sequence and the blocked 3′ end to the probe fragments,        wherein repeated thermal cycling is performed as described above        to preferentially extend the 3′ end of the second strand cDNA        probe copies with a second universal linker sequence, to form        double-linker probes from each probe fragment suitable for PCR        amplification, labeling and application to expression assays,        particularly oligonucleotide-based arrays.

Other embodiments of the Mini-WRAP-Probes methods employ these sameprocedures and reagents to create universal linkers on fragmented probesor fragmented DNA thus enabling either the amplification of the probesor fragments or the use of GeneTAGs and other reporters to increaseprobe signaling from small fragments.

One such application is the identification of small DNA fragments. DNAin preserved or frozen tissues, such as clinical, pathological orforensic specimens, are commonly degraded making it difficult toextract, amplify and identify the sequences. DNA fragments also appearin clinical specimens of blood and bodily fluids that are importantindicators of disease or cancer but are difficult to concentrate oridentify. Identifying degraded DNA is a particularly acute problem instudying Ancient DNA samples such as Egyptian and Etruscan mummies orbiological samples preserved in glaciers, bogs, amber, etc. The presentinvention provides additional modifications of the Mini-WRAP-Probemethod to amplify and identify any DNA fragment or set of fragments,wherein the following steps are applied to make and analyze the sample:

-   -   a. providing a sample of unknown DNA fragments;    -   b. applying a random probe modifier to the 3′ end of the        fragments to append a common universal linker, the random probe        modifier selected from the group consisting of the random        adapter composition and the random extender composition;    -   c. polymerizing a second strand cDNA copy of the fragments with        a primer comprising the universal linker sequence;    -   d. applying the random extender composition, further comprising        the same universal linker sequence and the blocked 3′ end, to        the fragments with repeated thermal cycling to preferentially        extend the 3′ end of the second strand cDNA copies with a second        universal linker sequence, to form double-linker fragments        suitable for PCR amplification; and    -   e. amplifying the fragments and sequencing them to determine        their sequence identity.

In this method, the probe modifier used to append the first universallinker is less critical, since there is only one potential 3′ target foreach probe fragment. Thus for this first step, the random adapter is thesimplest approach. However, once the second strand copies of thefragments are polymerized, the random extender composition provides animportant advantage since it will favor extending a 3′ end which lacks auniversal linker. Once a universal linker has been applied to a 3′ end,another random extender attempting to anneal to that end willpreferential bind the universal linker end of the random extender to thematching universal linker sequence already there—thus inactivating thatextender molecule for that cycle. Consequently, the random extendercomposition will preferentially apply only one universal linker to each3′ end.

Other embodiments of the Mini-WRAP-Probes methods enable improvedsensitivity with tissue microarrays or RNA arrays, wherein a cDNA probeprepared for such applications are modified by the above methods toappend universal linkers to the probes, wherein the steps to make theprobes comprise the following steps:

-   -   a. providing a fragmented cDNA probe;    -   b. applying a random probe modifier to append a first universal        linker to the 3′ end of the fragments;    -   c. polymerizing a second strand cDNA copy of the fragments;    -   d. applying the random extender composition, further comprising        the same universal linker sequence and the blocked 3′ end, to        the fragments with repeated thermal cycling to preferentially        extend the 3′ end of the second strand cDNA copies with a second        universal linker sequence, to form double-linker probe        fragments;    -   e. hybridizing the probes to an array of RNA targets;    -   f. hybridizing reporter units to the linkers of the probes, the        reporter units selected from the group of linker-primer-reporter        compositions, multi-linkers, and reporters, the reporter        comprising linear segments of label DNA or joined        polynucleotides with a single stranded universal linker end; and    -   g. detecting the reporter units to detect the RNA targets.

The purpose of this method is to maximize signaling by creating afractured set of probes, each of which can be labeled internally duringPCR amplification or by binding reporters to the universal linkers ofthe probes, wherein such reporters could include multi-linkers, GeneTAGsand GeneTAG arrays.

A very important need of molecular biology and drug discovery researchis the necessity of determining the sequences at the 5′ end of genetranscripts which are frequently under-represented or lost in commonprocedures (some of these procedures are called 5′ RACE). Suchinformation is needed to determine the functional full-length sequencesof a gene for drug discovery and patenting issues. The present inventionprovides a modified Mini-WRAP-Probe method wherein the Random Extenderand the Back-Tagging procedure are employed to find and duplicate theabsolute 3′ end of first strand cDNA copies of a specific gene, whereinthe steps of this procedure comprise the steps of:

-   -   i) providing a set of mRNA transcripts wherein the 5′ end of the        gene of interest has been copied as first strand antisense cDNA        by reverse transcriptase using a gene specific primer, wherein        the gene specific primer additionally comprises a universal        linker sequence and a capture moiety;    -   ii) capturing and purifying the first strand cDNA copies of the        targeted transcript;    -   iii) applying the random extender composition with rapid thermal        cycling to extend the 3′ end of the cDNA product with a        universal linker sequence, wherein a double-linker product is        formed suitable for PCR amplification; and    -   iv) amplifying and sequencing the double-linker cDNA product to        determine the sequences of the 5′ end of the gene.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, J. et al., Molecular Cloning; A Laboratory Manual, SecondEdition (1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984) APractical Guide to Molecular Cloning (B. Perbal, 1984); and the seriesMethods in Enzymology (Academic Press, Inc.).

The synthesis of some of the probe and reporter components of thepresent invention may be accomplished by conventional polymerase chainreaction (PCR) process. The protocol for PCR is set forth in Saiki etal., Science 230: 1350 (1985) and U.S. Pat. Nos. 4,683,195 and4,683,202. A PCR adapter-linker method is set forth in Saunders et al.(1990); Johnson (1990) and PCT 90/00434. Another PCR method employing amixture of primers is described in Meltzer et al., Nature-Genetics, 1(1): 24-28 (April 1992).

Probe and reporter components of the present invention are alsosynthesized by conventional methods on a commercially availableautomated DNA synthesizer, e.g. an Applied Biosystems (Foster City,Calif.) model 380B, 392 or 394 DNA/RNA synthesizer. Preferably,phosphoramidite chemistry is employed according to, e.g., Beaucage etal., Tetrahedron, 48:2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679. In preferredembodiments of the present invention, the probe has a nuclease resistantbackbone. Many types of modified oligonucleotides are available thatconfer nuclease resistance, e.g. phosphorothioate, phosphorodithioate,phosphoramidate. For phosphorothioates, see, e.g., Stec et al., U.S.Pat. No. 5,151,510; Hirschbein, U.S. Pat. No. 5,166,387; or Bergot, U.S.Pat. No. 5,183,885. For phosphoramidates, see, e.g., Froehler et al.,International application PCT/US90/03138. In some embodiments it may bedesirable to employ P-chiral linkages, e.g., Stec et al, EPO 92301950.9.

In several embodiments of the present invention, modifiedoligonucleotides are synthesized with internal spacers, commonlycomposed of carbon chains, which separate different functional regionsof the oligonucleotide. Generally, spacers derived from phosphoramiditeprecursors, such as the carbon chain Spacer Phosphoramidites C9 or C18from Glen Research, Inc. (Sterling, Va.), are preferred so that themodified oligonucleotides of the invention can be convenientlysynthesized with commercial automated DNA synthesizers, e.g. AppliedBiosystems, Inc. (Foster City, Calif.) model 394.

Spacer length may vary significantly depending on the nature of theprobe and primer sequence. Preferably, spacer moieties are synthesizedusing conventional phosphoramidite and/or hydrogen phosphonatechemistries. Several phosphoramidite or hydrogen phosphonate monomerssuitable for use in the present invention are set forth in Newton etal., Nucleic Acid Research, 21:1155-1162 (1993); Griffin et al., J. Am.Chem. Sot., 114:7976-7982 (1992) Jaschke et al., Tetrahedron Letters,34:301-304 (1992); Ma et al, International application PCT/CA92/00423;Zon et al., International application PCT/US90/06630; Durand et al.,Nucleic Acids Research, 18:6353-6359 (1990); and Salunkhe et al., J. Am.Chem. Soc., 114:8768-8772 (1992).

There is extensive background literature relating to the selection ofhybridization conditions, labeling procedures, and the like, which isapplicable to the principles and practice of the present invention. See,e.g. Wallace et al. Nucleic Acids Research 6:3543-3557 (1979); Crotherset al., J. Mol. Biol. 9:1-9 (1964); Gotoh, Adv. Biophys. 16:1-52 (1983)Wetruer, Critical Reviews in Biochemistry and Molecular Biology26:227-259 (1991); Breslauer et al., Proc. Natl. Acad. Sci. 83:374-3750(1986); Wolf et al., Nucleic Acids Research, 15:2911-2926 (1987); McGrawet al., Biotechniques, 8:674-678 (1990).

Conditions for annealing DNA based probes to DNA or RNA targets are wellknown, e.g., Nucleic Acid Hybridization, A Practical Approach (B. D.Homes, eds.), IRL Press, Washington, D.C. (1985). In general, whethersuch annealing or hybridization takes place is influenced by the lengthof the probes and the test substances, the pH, the temperature, theconcentration of mono- and divalent cations, the proportion of G and Cnucleotides in the hybridizing region, the viscosity of the medium andthe possible presence of denaturants. Such variables also influence thetime required for hybridization. The preferred conditions will thereforedepend upon the particular application. Such conditions, however, can beroutinely determined without undue experimentation.

For the joining of Adapters to probes, the preferred linking agent is aligase, such as T4 DNA ligase, using well-known procedures (Maniatis, T.in Molecular Cloning, Cold Spring Harbor Laboratory (1982)). Other DNAligases are also suitable. T4 DNA ligase may also be used when the testsubstance is RNA [Engler, M. J. et al., The Enzymes, Vol. 15, pp. 16-17(1982), Higgins, N. P. et al., Methods in Enzymology, Vol. 68, pp. 54-56(1979)]. Ligases from thermophilic organisms, e.g. Tth DNA ligase, Gene,Vol. 109, pp. 1-11(1991), New England Biolabs, (Beverly, Mass.), andAmpligase, Epcentre Technologies, Inc. (Madison, Wisc.) are preferred,so that ligation at higher temperatures may be carried out. Theligation, however, need not be an enzyme and, accordingly, the linkingagent may be a chemical agent which will cause the probe components tolink together. The invention is described using T4 DNA ligase as thelinking agent. This enzyme requires the presence of a phosphate group onthe 5′ end of one polynucleotide and a 3′ OH group on the neighboringpolynucleotide.

For covalent joining of probe or reporter components of the presentinvention, the preferred cross linking agent is a bi- or tri-functionalpsoralen compound such as 4,5′, 8-trimethylpsoralen which intercalatesthe bases of hybridized DNA strands and causes covalent cross linkingbetween them when treated with long wave ultraviolet light, preferablyin the range of 312 to 360 nanometers. Site specific cross-linking canalso be facilitated by synthesizing an oligonucleotide probe componentwith a terminal psoralen molecule tethered by a carbon chain. Commercialreagents, such as C2 psoralen and C6 psoralen from Glen Research, Inc.(San Diego, Calif.), allow the termination of a syntheticoligonucleotide with an attached psoralen suitable for inducingcrosslinking with double or triple strand configurations, respectively,using standard phosphoramidite chemistry on a automated DNA synthesizer,e.g. Applied Biosystems, Inc. (Foster City, Calif.) model 394. Thedurability of complementary hybridization between probe and reportercomponents may also be increased by employing artificial nucleotides;e.g. pdC-CE, pdU-CE, 5-Me-dC, Glen Research, Inc. (Sterling, Va.), whichcan significantly raise melt temperature (Tm) by several degrees, andcan diminish non-specific binding of these components.

The probe and reporter molecules of this invention can be labeled duringPCR amplification in the presence of appropriately modified dNTPs, orthey can be labeled after completion of the PCR reaction by chemical orenzymatic modification of the PCR products. When the reporters areconstructed of synthetic oligonucleotides, they can be labeled directlyor indirectly by incorporating modified bases that either carry labelingagents or provide chemical or immunological means for the attachment oflabeling agents. Alternatively, such reporters may contain secondarylinkers for binding short oligonucleotides that are conjugated tolabeling agents—usually at one end.

Any of the various labeling techniques, direct or indirect, may be usedto label probes or reporters, including but not limited to fluorescentchemicals, radioactive materials, chemical haptens, or enzymaticmodifiers. More than one label can be used. Preferred modified dNTPsinclude but are not limited to Cy3 or Cy5 labeled derivatives of dUTP ordCTP, biotin-16-dUTP; digoxigenin-11-dUTP; biotin derivatives of dATP;fluoresceinated-dUTP; rhodamine labeled derivatives of dUTP or dCTP;hydroxy coumarin-labeled derivatives of dUTP; resorufin-11-2′-dUTP, andthiol or amine modified dNTPs, e.g. amino-allyl-dUTP, Sigma Chemical Co.(St. Louis, Mo.), Amino-Modifier C6-dT, Glen Research, Inc. (Sterling,Va.). Other potential labels that may be attached or conjugated to probeor reporter components include but are not limited to: (1) gold andsilver particles; e.g. monomaleimido Nanogold, LI Silver, etc.,Nanoprobes, Inc., (Stony Brook, N.Y.); Colloidal Gold, Sigma ChemicalCo. (Saint Louis, Mo.); (2) chemiluminescent or bioluminescent moleculessuch as aequorin, e.g. Aqualite, Sealite Sciences, Inc., (Norcross,Ga.); and (3) agents which can provide Raman spectrometry signaling suchas DNA and histological dyes; e.g. Methyl green, Cresyl fast violet,Acridine orange, Ponceus S, Malachite green oxalate, Luxol fast blue,Cresyl violet acetate and Bromophenol blue; double and or triple bondedchemical labels; e.g. Chloracetonitrile, Propargyl chloride,3′Methoxybenzyl chloride and alpha Bromo p-tolunitrile, Aldrich ChemicalCompany, Inc. (Milwaukee, Wisc.); and propyne or methyl modifiedphosphoramidite nucleosides; e.g. pdC-CE, pdU-CE, 5-Me-dC, GlenResearch, Inc. (Sterling, Va.). The staining intensity achieved withprobes or reporters may be amplified with a variety of systems,including but not limited to fluorochrome conjugated avidin and/orlabeled antibodies. Similarly, other known detection schemes such aslabeling of probe molecules with enzymes, sulfur or mercury may beapplied in order to suit special experimental conditions.

Methods for introducing oligonucleotide functionalizing reagents or tointroduce one or more sulfhydryl, amino or hydroxyl moieties into probeor reporter sequences are described in U.S. Pat. No. 4,914,210. Suchmodified nucleotides can provide multiple signaling sites byincorporating them along the length of the probe or reporter molecule orat the ends of attached oligonucleotides. A 5′ phosphate group can beintroduced as a radioisotope by using polynucleotide kinase and gamma32P-ATP to provide a reporter group. Biotin can be added to the 5′ endby reacting an aminothymidine residue, or a 6-amino hexyl residue,introduced during synthesis, with an N-hydroxysuccinimide ester ofbiotin. Labels at the 3′ terminus may employ polynucleotide terminaltransferase to add the desired moiety, such as for example, cordycepin35S-dATP, and biotinylated dUTP.

The present invention provides and contemplates the combination of thenovel compositions of matters describe above, such as the Modifiedpoly-T primers the various adapters and extenders, and the probe setcompositions, wherein different variations may be created that are notspecifically describe herein. These potential variations andcombinations include modifications of the WRAP-Probe,Amplified-WRAP-Probe, and Mini-WRAP-Probe methods and their variousmanifestations thereof, with various GeneTAG, TinkerTAG or ChipTAGsignal amplification systems. Alternatively, the compositions of matterand methods of the present invention are contemplated to be employed incombination with other commercial probe and signaling systems such asthe dendrimers of Polyprobe, Inc. (Media, Penn.) [U.S. Pat. No.5,487,973] and the branch DNA (bDNA) components of Chiron Corp.(Emeryville, Calif.) [U.S. Pat. No. 5,124,246].

The probes and reporters of the present invention can be employed asdiagnostic or drug discovery assays for a wide range of biomedicalsamples, including detection of nucleic acids and gene expressionprofiles in human diagnostics, forensics, and genomic analyses. See,e.g., Schena et al., Science, 270: 467-470 (1995); Schena, et al., Proc.Natl. Acad. Sci., 93:10614-9 (1996); Shalon et al., Genome Res., 6:639-45 (1996); DeRisi et al., Nature Genetics, 14: 457-60, (1996);Heller et al., Proc. Natl. Acad. Sci., 94: 2150-5, (1997); Khan et al.,Cancer Res., 58: 5009-13 (1998); Khan et al., Electrophoresis, 20: 223-9(1999); Caskey, Science 236:1223-1228 (1987); Landegren et al. Science,242:229 237 (1988); and Arnheim et al., Ann. Rev. Biochem., 61:131-156(1992). Other diagnostic applications of the present invention includesamples from the environment, e.g. from public water supplies, samplesfrom foodstuffs, and from other biological or clinical samples, such asblood, saliva, lung sputum, semen, buccal smears, urine or fecal waste,cell and tissue biopsies and micro dissections, amniotic fluid, ortissue homogenates of plants, animals, or human patients, and the like.

The compositions and methods of the present invention can be readilyemployed in a variety of membrane formats such as expression macro andmicroarrays, dot blots, and Northern blots; in gels such as agar orpolyacrylamide; in a variety of in situ formats to detect or map genesor RNA transcripts in sectioned tissue and tissue microarrays; incultures or microwell plates to detect infectious microorganisms orunbound DNA fragments extracted from bodily fluids or wastes; and invarious solid substrate chip formats that detect genes, mutations ormRNA expression levels, including but not limited to oligonucleotidemicroarrays, cDNA microarrays, and molecular detection chips employingfluorescence, radioactivity, optical interferometry, Raman spectometryor semi-conductor electronics.

EXAMPLES Example 1 Sample Molecular Compositions of the PresentInvention

Universal GeneTAG Linkers:

1. Red 5′CTACGATACGATAGCGCCTAAGAGTAG (Seq. ID. No. 1) and itscomplement.

2. Green 5′CCTAGACCTACGACATAGGTACCCTAC (Seq. ID. No. 2) and itscomplement.

3. Blue 5′CGTAGAACTAGCACGCTACGTACTAGG (Seq. ID. No. 3) and itscomplement.

4. Orange 5′GGCTATCGCTACGTAGACTAGACCTAC (Seq. ID. No. 4) and itscomplement.

Modified Poly-T Primer with red or green universal linker and anchorend:

(Seq. ID. No. 1, 5) 1. 5′CTACGATACGATAGCGCCTAAGAGTAG-TTTTTTTTTTTTTTTVN(Seq. ID. No. 2, 5) 2. 5′CCTAGACCTACGACATAGGTACCCTAC-TTTTTTTTTTTTTTTVN

Double-Linker WRAP-Probe Set 1: Showing one probe strand with Red andBlue Universal Linkers, and with the variable target sequence indicatedby S1 . . . Sn

(Seq. ID. No. 1, 6) 5′CTACGATACGATAGGGCCTAAGAGTAG-S1 . . . Sn-CCTAGTACGTAGCGTGCTAGTTCTACG

Double-Linker WRAP-Probe Set 2: Showing one probe strand with Green andOrange Universal Linkers, and with the variable target sequenceindicated by S1 . . . Sn

(Seq. ID. No. 2, 7) 5′CCTAGACCTACGACATAGGTACCCTAC-S1 . . . Sn-GTAGGTCTAGTCTACGTAGCGATAGCC

Specific Adapter:

Version with Blue Universal Linker: a first polynucleotide with bluelinker, an overhang specific to a restriction enzyme cut site (indicatedby S1 . . . Sn), and a second complementary polynucleotide preferably5′phosphorylated:

(Seq. ID. No. 3, 6) 5′CGTAGAACTAGCACGCTACGTACTAGG-S1 . . . Sn5′P-CCTAGTACGTAGCGTGCTAGTTCTACG

Version with Orange Universal Linker and Label:

(Seq. ID. No. 4, 7) 5′GGCTATCGCTACGTAGACTAGACCTAC-S1 . . . Sn5′P-GTAGGTCTAGTCTACGTAGCGATAGCC-LABEL

Random Adapter: Version with Blue Universal Linker: a firstpolynucleotide with blue linker, a random overhang sequence, typicallyof 2N's (indicated by N1 . . . Nn), and a second complementarypolynucleotide preferably 5′ phosphorylated:

(Seq. ID. No. 3, 6) 5′CGTAGAACTAGCACGCTACGTACTAGG-N1 . . . Nn5′P-CCTAGTACGTAGCGTGCTAGTTCTACG

Random End-Linker (random extender): Version with Blue Universal Linker:Showing a polynucleotide with Blue linker sequences, a random overhangsequence, typically of 6 to 9N's (indicated by N1 . . . Nn), and ablocked 3′ end. (Seq. ID. No. 3)

5′CTACGATACGATAGCGCCTAAGAGTAG-N1 . . . Nn-block

Homopolymeric Adapter: Version with Blue Universal Linker: Showing afirst polynucleotide with Blue linker sequences and a poly-C or poly-Gsequence (indicated by C1 . . . Cn), and a second polynucleotide whichis complementary to the first nucleotide and preferably 5′phosphorylated:

(Seq. ID. No. 3, 6) 5′CGTAGAACTAGCACGCTACGTACTAGG-C1 . . . Cn5′P-CCTAGTACGTAGCGTGCTAGTTCTACG

Homopolymeric extender: Version with Blue Universal Linker: Showing apolynucleotide with Blue linker sequences and a poly-C or poly-Gsequence (indicated by C1 . . . Cn): (Seq. ID. No. 3)

5′CGTAGAACTAGCACGCTACGTACTAGG-C1 . . . Cn

Labeled ChipTAG Primers:

Red and Blue Linker/Primers with Cy5 Fluor.

Red 5′-cy5-CTACGATACGATAGCGCCTAAGAGTAG (Seq. ID. No. 1) Blue5′-cy5-CGTAGAACTAGCACGCTACGTACTAGG (Seq. ID. No. 3)

Green and Orange Linker/Primers with Cy3 Fluor.

Green 5′-cy3-CCTAGACCTACGACATAGGTACCCTAC (Seq. ID. No. 2) Orange5′-cy3-GGCTATCGCTACGTAGACTAGACCTAC (Seq. ID. No. 4)

Example 2 One-Linker WRAP-Probe Method

Total RNA is extracted from A549 lung cancer cells by standard methods.Reverse transcriptase (RT) is then employed to copy the mRNA transcriptsto cDNA using a Modified poly-T Primer known as R-GT-RTP (Seq. ID. No.8) having a 3′ end of 15 poly-T's and a 5′ end with a universal linkersequence that is similar to but differing in part from the Red UniversalLinker of Example 1. For a comparative sample an alternative Modifiedpoly-T Primer, known as G-GT-RTP (Seq. ID. No. 9) is used for the RTreaction to provide a second universal linker sequence wherein thosesequences are also similar to but differing in part from the Greenuniversal linker of Example 1.

The Examples 2 through 7 use these earlier versions of the red and greenuniversal linkers in all their products, and thus the text of thoseExamples identifies them differently with the terms First-RED andFirst-GREEN in the descriptions to identify these sequence differences.

For a 25 ul reaction, 40 micrograms of A549 RNA was combined with 2 ulof 100 picomol/ul R-GT-RTP (Seq. ID. No. 8), 10 × PCR buffer II, 25 mmMgCl2, 1 ul each of 10 mM dATP, dGTP and dTTP, 6 ul of 1 mM dCTP, 3 mlof 1 mM Cy3 dCTP (AP Biotech), and dH2O, and the mixture was placed in a70 degrees C. waterbath for 10 min and allowed to cool at roomtemperature for 5 min. Then 50 units of MuVL reverse transcriptaseenzyme (Perkin Elmer) was added along with 20 units of RNase inhibitor(Perkin Elmer) and the mixture was incubated at 42 degrees C. for 1hour. The product was purified with a Centri-Sep spin column (PrincetonSeparations).

Type I GeneTAGs with a First-RED linker on the proximal end and Type IIGeneTAGs with a First-GREEN linker on the proximal end were made andboth types were labeled “red” with Cy5-dCTP by PCR amplification of anarbitrary MTB template, 600 bp long, using 2 ul of 0.25 ug/ul oftemplate. The primers employed for Type I GeneTAGs are RR-SPC-F (SEQ IDNO. 10, 11) and GR-SPC-R: (SEQ ID NO. 12, 13) using 2 ul each at 10pmol/ul. The primers employed for Type II GeneTAGs are GR-SPC-F: (SEQ IDNO. 14, 11) and RR-SPC-R: (SEQ ID NO. 15, 13) using 2 ul each at 10pmol/ul. The internal spacers are identified as 99 indicating two C9phosphoramidite spacers (Glen Research). Fluorescent labeling isaccomplished during PCR amplification of the reporters whereinnucleotides are added with low dCTP (6 ul of 1 mM) plus normal dATP,dTTP and dGTP (1 ul each of 10 mM) plus 3 ul of 25 uM Cy5-dCTP. Taq, 10×buffer and dist. H2O were added and the mixture cycled 40 times at 94degrees C., 55 degrees C. and 72 degrees C. for about 1 min per step.The products were purified twice with a Centri-Sep spin column.

The probes were hybridized overnight at 65 degrees C. to cDNA chipsarrayed on poly-L-lysine coated glass slides with a Genetic Microsystemsspotter using 5 ul of probe mixed with 7 ul of hybridization buffer,said buffer consisting of 3.5×SSC and 0.2% SDS and containing Cot 1 DNA,poly-A RNA, and tRNA. Each gene location on these chips are duplicated 5times in vertical columns. After a brief wash with hybridization buffer,GeneTAGs were hybridized for an additional 2 hours under the sameconditions. The chips were gently washed for 5 min each in threesteps: 1) 2×SSC, 0.1% SDS, 2) 1×SSC, and 3) 0.1×SSC.

GeneTAG Modified Poly-T Primers: a) First-RED Linker version R-GT-RTP5′CTACGATACGATAGGGCCTAAGAGTAG-TTTTTTTTTTTTTTT (Seq. ID. No. 8) b)First-GREEN Linker version G-GT-RTP5′GCCTAGACCTAGGGGTAGCTAGGCTAC-TTTTTTTTTTTTTTT (Seq. ID. No.9) Type IGeneTAG Spacer Oligomers: a) Proximal Spacer Oligomer RR-SPC-F:5′CTACTCTTAGGCCCTATCGTATCGTAG- (SEQ ID NO. 10, 11)-99—CCAGGGTTTTCCCAGTCACGAC b) Distal Spacer Oligomer GR-SPC-R:5′GCCTAGACCTAGGGGTAGCTAGGCTAC- (SEQ ID NO. 12, 13)-99—GAGCGGATAACAATTTCACACAGG Type II GeneTAG Spacer Oligomers: a)Proximal Spacer Oligomer GR-SPC-F: 5′GTAGCCTAGCTACCCCTAGGTCTAGGC- (SEQID NO. 14, 11) -99—CCAGGGTTTTCCCAGTCACGAC b) Distal Spacer OligomerRR-SPC-R: 5′CTACGATACGATAGGGCCTAAGAGTAG- (SEQ ID NO. 15, 13)-99—GAGCGGATAAGAATTTCACACAGG

The chips are scanned with a Genetic Microsystems laser scanner andproduce two gene expression profiles from the “green” channel and the“red” channel showing, respectively, differential signaling with boththe labeled probes and with the labeled GeneTAGs bound to the probes.Since each gene target arrayed on these chips is duplicated in verticalcolumns five times, it is easy to see and confirm true differences ingene expression between genes. Approximately 20 gene locations on thechip show highly significant “green” labeling indicating specific geneexpression levels for these cells, and approximately 200 gene locationsshowed significant “red” labeling indicating additional gene expressionlabeling provided by the GeneTAGs bound to the probes. Labelingintensity varies per each vertical set of gene targets for both thegreen and red channels indicating gene expression monitoring. See FIGS.6A and B.

Example 3 Double-Linker WRAP-Probe Method with Restriction Cutting andAdapter Ligation

Total RNA was extracted from A549 lung cancer cells by standard methods.A 40 ug sample was treated with reverse transcriptase (RT) and aModified Poly-T Primer to make ds cDNA copies of the mRNAs with a firstlinker, to cut and capture the end fragments, and to add a second linkerby ligating an adapter. The following steps were employed to make theprobes and perform a chip analysis:

1. Full length first strand cDNAs were made with one hour exposure toMuVL RT (Gibco) at 37 degrees C. using a Modified Poly-T Primer (Seq.ID. No. 16) having a 5′ biotin capture moiety, an overlap linkersequence and a poly-T segment. Nucleotides including Cy3-dCTP (APBiotech) were incorporated as described above to provide “green”labeling.

2. Double stranded cDNA was made with E. coli DNA polymerase I, Rnase Hand DNA ligase (Gibco kit) with a two hour exposure at 16 degrees C.

3. The ds cDNAs were treated with the restriction enzyme N1a III (NewEngland Nuclear) for 7 hours at 37 degrees C. and purified twice withCenti-Sep spin columns.

4. The end fragments were captured with 10 mg strepavidin-coatedmagnetic beads (Dynal).

5. A pre-annealed First-RED Specific Adapter (Seq. ID. No. 17, 18) wasprepared and ligated to the fragments with T4 DNA ligase (BoehringerMannheim) for 30 min. at 37 degrees C. providing a first 5′ First-REDLinker sequence.

6. The adapter modified fragments were again captured on magnetic beads,denatured with 0.2 M NaOH, and the eluted probes retained andneutralized.

7a. In part of the sample, a two part overlap linker (Seq. ID. No. 19)was annealed and cross linked to the ss probes to form a second 5′First-RED-Linker. Such probes are double-linker WRAP probes.

7b. Another part of the sample was instead used as templates foramplifying and labeling the probes by PCR. In this case, amplificationwas initially accomplished with a set of primers consisting of a firstprimer, which is the First-RED version of GeneTAG Modified Poly-T Primerused above (Seq. ID. No. 8), (this binds to the poly-A segment of thesense cDNA), and of a second primer, which is the First-REDLinker-primer (Seq. ID. No. 20) which contains the same sequences as the5′ end of the first primer. PCR is conducted for 10 cycles at 94 degrees30′, 48 degrees 30′ and 72 degrees 45′. Then PCR amplification isrepeated for 30 cycles using only the GeneTAG First-RED primer.Alternatively, in step 5 above a pre-annealed First-GREEN SpecificAdapter (Seq. ID. No. 21, 22) could be employed for ligation to the cutsites, and thus, the resulting probes could be PCR amplified with theFirst-GREEN Modified Poly-T Primer (Seq. ID. No. 9). and a First-GREENprimer (Seq. ID. No. 23) in the same manner as described above using theFirst-RED compositions. Furthermore, the above GeneTAG First-GREEN orFirst-RED compositions could be employed together such that one primerwould employ the First-GREEN linker/primer sequence and the other wouldemploy the First-RED linker/primer sequence.

The resulting WRAP-Probes from step 7b. above were hybridized to cDNAchips overnight and then treated for 2 hours with Type III GeneTAGspreviously labeled “red” with Cy5-dCTP (AP Biotech) as described inExample 2. Type III GeneTAGs have a proximal linker which binds to theFirst-RED Linker sequences of the WRAP-Probes and two distal linkersthat bind to the proximal linker of Type IV GeneTAGs. The chips arewashed once and then Type IV GeneTAGs are applied for two additionalhours. The chips are then washed three times as described in Example 2above. These chips are similarly prepared with vertical duplications ofdifferent gene targets, but in this case, four sets of targets vs. fivesets of targets are represented in the vertical columns. The chips arescanned with a Genetic Microsystems scanner showing differentiallabeling with the probes and the GeneTAGs as described above. See FIGS.7 and 8. This approach produces more extensive labeling with two layersof GeneTAGs.

GeneTAG Components: Modified RT primer5′biotin-CGACTACCTATCTAC-TTTTTTTTTTTTTTT (Seq. ID. No. 16) First-REDGeneTAG Adapter part 1: 5′CTACGATACGATAGGGCCTAAGAGTAG-CATG (Seq. ID. No.17) First-RED GeneTAG Adapter part 2: 5′CTACTCTTAGGCCCTATCGTATCGTAG(Seq. ID. No. 18) Overlap Linker5′CTACGATACGATAGGGCCTAAGAGTAG-CGACTACCTATCTAC (Seq. ID. No. 19) GeneTAGFirst-RED Primer: 5′CTACGATACGATAGGGCCTAAGAGTAG (Seq. ID. No. 20)First-GREEN Specific Adapter part 1: 5′GCCTAGACCTAGGGGTAGCTAGGCTAC-CATG(Seq. ID. No. 21) First-GREEN Specific Adapter part 2:5′GTAGCCTAGCTACCCCTAGGTCTAGGC (Seq. ID. No. 22) GeneTAG First-GREENPrimer: 5′GCCTAGACCTAGGGGTAGCTAGGCTAC (Seq. ID. No. 23) b) First-GREENLinker version G-GT-RTP 5′GCCTAGACCTAGGGGTAGCTAGGCTAC-TTTTTTTTTTTTTTT(Seq. ID. No. 9) Type III GeneTAG Spacer Oligomers: a) Proximal SpacerOligomer RR-SPC-F: 5′CTACTCTTAGGCCCTATCGTATGGTAG- (SEQ ID NO. 10, 11)-99—CCAGGGTTTTCCCAGTCACGAC b) Distal Spacer Oligomer GR-SPC-R:5′GCCTAGACCTAGGGGTAGCTAGGCTAC- (SEQ ID NO. 12, 12, 13)-GCCTAGACCTAGGGGTAGCTAGGCTAC- -99—GAGCGGATAACAATTTCACACAGG Type IVGeneTAG Spacer Oligomers: a) Proximal Spacer Oligomer GR-SPC-F:5′GTAGCCTAGCTACCCCTAGGTCTAGGC- (SEQ ID NO. 14, 11)-99—CCAGGGTTTTCCCAGTCACGAC b) Distal Spacer Oligomer RR-SPC-R:5′CTACGATACGATAGGGCCTAAGAGTAG- (SEQ ID NO. 15, 15, 13)-CTACGATACGATAGGGCCTAAGAGTAG- -99—GAGGGGATAACAATTTCACACAGG

Example 4 WRAP-Probe Method with Restriction Cutting, Adapter Ligationand ChipTAG Labeling

A 10 microliter probe sample from Step 7b. of Example 3 above wasre-amplified by PCR and applied to chips as described in Examples 2 and3 above. However, in this case the First-RED ChipTAG primer (Seq. ID.No. 24) was employed as a single primer to globally amplify all probeproducts. Furthermore, internal labeling was not employed, and thusbound labeling was achieved from a single Cy5 fluor being attached tothe 5′ end of each single stranded probe component. Additionally, afterPCR and before hybridization was conducted, an additional 0.2 microliteraliquot of First-RED ChipTAG primer at a concentration of 100picomoles/microliter was added to the sample of purified probes. Sincethese added primers are labeled and they are capable of binding to the3′ linker end of each bound probe, they can provide additional signalingper probe. For two color comparisons, an additional ChipTAG with theFirst-GREEN sequences (Seq. ID. No. 25) can be employed to provide PCRamplified probes with a single Cy3 label per amplified product. Thesechips also used vertical duplications of the gene targets arrayed, andin this case, four duplications were again employed for each column oftargets. See FIG. 9.

First-RED ChipTAG Primer (Seq. ID. No. 24)5′ Cy5-CTACGATAGGATAGGGCCTAAGAGTAG First-GREEN ChipTAG Primer (Seq. ID.No. 25) 5′ Cy3-GCCTAGACCTAGGGGTAGCTAGGCTAC

Example 5 WRAP-Probe Method with Short RT and Random End-Linker

Step 1: Samples of poly-A mRNA from mouse liver of controls and ANT-1knockouts were employed to prepare probes for expression microarrays orchips. In each case 50 nanograms of poly-A mRNA were treated withGeneTAG RT primers in a brief RT reaction of 30 seconds using 1 ug ofG-GT-RTP at 100 pmol/ul added to a 20 ul reaction. These RT reactionsused Superscript II RT and 5×RT buffer (Gibco kit), dNTPs, and 0.1M DTTand no labeling reagents. First the template and primer were combined inH2O for 5 min at 72 degrees C. and then placed on ice for 10 min.Thereafter, the enzyme, buffer, dNTPs and DTT were added, maintained at42 degrees C. for 30 sec, and the reaction was stopped again on ice. Theproduct was treated for 15 min at 75 degrees C. to inactivate theenzyme. The products were purified by Centri-Sep spin columns. Thissub-procedure adds a first linker/primer site to the 5′ ends of the cDNAprobes and terminates RT copying prematurely to normalize the lengths ofthe probes.

Step 2: The above Control and ANT-1 samples were added to separate 30 ulreactions containing 100 picomoles of the First-GREEN version GeneTAGRandom End-Linker (Seq. ID. No. 26), plus 10×PCR buffer, dNTPs, Taqpolymerase and dH2O. The GeneTAG End-Linkers are 3′ modified to preventforward copying. Because of this modification, they are only effectivein this reaction if they bind partially to the 3′ end of the probes viathe random segment and serve as a template to back extend the probeswith a GeneTAG linker/primer sequence. PCR thermal cycling is thenperformed at 94 degrees C. for 10 sec, 42 degrees C. for 30 sec, and 72degrees for 10 sec, for a total of 198 cycles. The products are purifiedwith spin columns. This sub-procedure commonly extends the 3′ ends ofthe probes with the First-GREEN GeneTAG linker sequence providing asecond linker/primer site. In alternate preparations, the First-REDversion GeneTAG End-Linker (Seq. ID. No. 27) is employed to put aFirst-RED GeneTAG linker sequence on the 3′ end. The N's listed in theEnd-Linker sequences below indicate bases which are randomlyincorporated during oligonucleotide synthesis as either A, T, G, or C.

Step 3: The above Control and ANT-1 samples are again subjected to PCRcycling, but this time with conditions allowing the exponentialamplification and labeling of the probes using the double-linker sitesas primer sites. The samples are added to 100 ul PCR reactionscontaining 100 picomoles of the GeneTAG First-GREEN primer (Seq. ID. No.23), 10×PCR buffer, Taq polymerase, and 16 ul of a mix of dNTPs withamino-allyl dUTP (Sigma) [wherein the stock mix contains 4 ul 100 mMAA-dUTP, 6 ul 100 mM dTTP, 10 ul 100 mM dATP, 10 ul 100 mM dCTP, 10 ul100 mM dGTP and 760 ul dH2O]. PCR thermal-cycling is performed at 94degrees C. for 30 sec, 48 degrees C. for 1 min, and 72 degrees for 30sec, for a total of 40 cycles.

The probes are purified with Microcon-30 columns and dried in aSpeedVAC. The amino-conjugated bases of the Control probes are thencoupled to Cy3 dye and the amino-conjugated bases of the ANT-1 probesare coupled to Cy5 dye using Cy3 or Cy5 monofunctional reactive dyepacks from APBiotech. Before mixing the samples, the reactions arequenched with hydroxylamine to prevent cross coupling. Unincorporated orquenched Cy dyes are removed by purification with QiaQuick columns(Qiagen) and the labeled probes are concentrated by drying with aSpeedVAC. The probes were combined and one fifth the resulting samplewas hybridized to mouse expression microarrays at 65 degrees C. for 16hrs in 3.5×SSC plus 2% SDS and washed as described above. This samplingis essentially equivalent to starting with 10 nanograms of poly-A mRNAper sample. This procedure gave very short probes with limited chipsignaling suggesting the need to reduce hybridization temperature andincrease RT timing.

“First-GREEN” GeneTAG Random End-Linker (Seq. ID. No. 26)5′GCCTAGACCTAGGGGTAGCTAGGCTAC-NNNNNNNNN-99 “First-RED” GeneTAG RandomEnd-Linker (Seq. ID. No. 27) 5′CTACGATACGATAGGGCCTAAGAGTAG-NNNNNNNNN-99

Example 6 WRAP-Probe Method with Short RT and Random End-Linker(Membrane Arrays)

The Short RT and Random End-Linker method was more effective with longerRT extension periods. The following examples were prepared fromexperiments with human monocytes (derived from Red Cross buffy coatpreps) to compare Control monocytes and IL-13 Treated monocytes.

Step 1: Essentially the same procedures from Step 1 of Example 5 abovewere employed except that the starting samples consisted of 1 microgramof total RNA per sample and the RT reaction for the Control RNA used theFirst-GREEN Modified Poly-T Primer (Seq. ID. No. 9) while the RTreaction for the IL-13 Treated RNA used the First-RED Modified Poly-TPrimer (Seq. ID. No. 8). The RT reactions of 20 microliters contained100 picomoles of GeneTAG Modified Poly-T Primer, 1 ul RT enzyme(SuperScript II) and 4 ul 5× buffer (Gibco kit), 1 ul dNTPs, 2 ul 0.1 MDTT and dH2O. The primers and RNA templates were mixed at 72 degrees C.for 5 min, and then the enzyme and other components were added andmaintained at 42 degrees C. for various Short RT times of either 2, 5,10 or 20 minutes, followed by 75 degree C. treatment for 15 min to stopthe cDNA copying reaction prematurely from all transcripts regardless ofgene specific differences in transcript length. The products werepurified with Bio-Spin P-30 chromatography columns (Bio-Rad).

Step 2: This multi-cycle step was performed essentially the same as inExample 5 above except that the Control samples employed the First-GREENRandom End-Linker (Seq. ID. No. 26) while the IL-13 Treated samplesemployed the First-RED Random End-Linker (Seq. ID. No. 27). Thus theControl probes would have First-GREEN linker/primer sites at both endswhile the IL-13 Treated probes would have First-RED linker/primer sitesat both ends.

Step 3: This step was performed essentially the same as in Example 5above except that the probes from the 20 min Short RT procedure werelabeled with P-32 dCTP vs. fluorescence, and furthermore, the Controlprobes were amplified by PCR using the GeneTAG First-GREEN primer (Seq.ID. No. 20), and the IL-13 Treated probes were PCR amplified with theGeneTAG First-RED primer (Seq. ID. No. 23). For 100 ul reactions, 30 ulof probe template was employed with 100 picomoles of First-GREEN orFirst-RED for a total of 30 PCR cycles. Both products were purified,counted and adjusted to yield probes with an activity of one millioncpm/ml.

Nylon membranes were arrayed with 10 gene target samples that werearranged in vertical columns of five slot blots per column. Eachmembrane of approximately 6 by 10 cm duplicated this 10 gene arraypattern twice in a side by side arrangement. Each dot contained 200nanograms each of plasmid cDNA from 6 candidate and 4 control targets:candidates: 5-LO, 12-LO, FLAP, COX-1, COX-2, 15-LO, controls: Leptin,TNF-alpha, yeast and h-Actin. The target samples were denatured with 0.1N NaOH, neutralized with Tris-HCl buffer, and UV crosslinked. Membraneswere prehybridized for 4 hours in rotating roller bottles with 20 ml ofhybridization solution (Rapid-hyb buffer, Amersham Life Science). Thelabeled and amplified probes were then added for overnight hybridizationat 48 degrees C. with the same solutions, and then they were washedsequentially with 2×SSC and 0.1% SDS for 15 min, 0.2×SSC for 15 min 2times, and 0.1×SSC for 15 min also at 48 degrees C. Expression profilingwas obtained by exposing x-ray film for 12 hours. The repeated patternsevident within membranes also differed slightly between control andIL-13 treated monocytes, and as expected, IL-13 treatment up-regulatedthe expression activity of 15-LO. See FIG. 10.

Example 7 Amplified WRAP-Probe Method with Short RT and Random Adapter(on Membrane Arrays)

The same samples prepared for Example 6 above were also employed for analternate method of attaching the second linker/primer sequence with aligated GeneTAG Adapter. In these experiments, the PCR extension timewas also increased from 30 sec to 1.5 min to allow more representationof the longer RT products in the sample of amplified probes. This changealso shifts the sampling that will appear on the chip. Since all priormethods for expression microarrays are biased in signaling relative toprobe length, further study is needed to determine which profilingpattern will prove to be more accurate.

Step 1: Essentially the same procedures from Step 1 of Example 6 wereemployed with starting samples consisting of 1 microgram of total RNAper sample. All samples were from monocyte controls and the RT reactionsused the First-GREEN Modified Poly-T Primer (Seq. ID. No. 9) to producethe first linker/primer site. Alternatively, the First-RED ModifiedPoly-T Primer (Seq. ID. No. 8) could be employed for other comparisons.Short RT was conducted as described above with reduced RT exposure timesof 2, 5, 10 or 20 minutes.

Step 2: This step was performed quite differently from that of Example 4above since, in this case, the second linker/primer site was affixed tothe 3′ ends of the probes by direct ligation of First-GREEN RandomAdapters. These random Adapters are composed of two oligonucleotidesthat are annealed together and of which one component provides a twobase overhang of random sequences. These Random Adapters consist of afirst oligonucleotide with First-GREEN linker sequences on the 5′ endand two N's on the 3′ end (Seq. ID. No. 28), and of a secondoligonucleotide (Seq. ID. No. 29) with sequences complementary to theFirst-GREEN linker sequences and with the 5′ end phosphorylated duringsynthesis to facilitate ligation. In alternate preparations, a First-REDversion of such Adapters could be employed which is made of a firstoligonucleotide with First-RED linker sequences on the 5′ end and twoN's on the 3′ end (Seq. ID. No. 30), and of a second oligonucleotide(Seq. ID. No. 31) with sequences complementary to the First-RED linkersequences. For this preparation, the two Random Adapter oligonucleotideswere mixed together at a concentration of 100 picomoles/ul per productand then annealed for 2 hours at 37 degrees C. with 10 percent 10×PCRbuffer and dH2O. This product was chilled on ice and stored at −20degrees C. Samples treated by Step 1 above were combined with saidRandom Adapters in a 30 ul reaction consisting of 20 ul of probetemplate, 1 ul of Adapter, then 2 ul of T4 ligase and 6 ul of 5×Ligation buffer (Gibco kit) and dH2O. This ligation was conducted at 16degrees C. overnight, but alternatively could be accomplished at 37degrees C. for one hour. This reaction joins the Random Adapter to the3′ end of the probes creating a second linker/primer site.

Step 3: The probes can be amplified and labeled by PCR with standardmethods. However, in this case two stages of amplification wereemployed. First, a 100 ul reaction of 20 cycles is conducted with 10 ulof probe product (after Adapter ligation), 1 ul of First-GREEN Linker(Seq. ID. No. 23) at 100 pmols/ul, 10 ul of 10×PCR buffer, 8 ul ofdNTPs, 1 ul of Taq polymerase and dH2O. Then 10 ul of the above reactionis again amplified in a second PCR reaction of 100 ul for 30 cyclesusing 5 ul of P-32 dCTP (NEN Dupont) plus 1 ul of 1 mM dCTP, and 1 uleach of 10 mM DATP, dTTP and dGTP. Both reactions are thermocycled at 94degrees C. 30 sec, 55 degrees C. 1 min, and 72 degrees C. for 1.5 min.The probes are purified by Centri-Sep spin column and applied to themembranes as described in Example 5 using pre-hybridization,hybridization and washing as indicated above. This procedure labels theprobes internally. See FIG. 11. These results show that 2 min, 5 min and10 min Short RT yield effective and relatively similar probe productsand expression profiles while 20 min Short RT yields a relatively weakerset of products.

First-GREEN Random Adapter part I (Seq. ID. No. 28)5′ GCCTAGACCTAGGGGTAGCTAGGCTAC-NN First-GREEN Random Adapter part 2(Seq. ID. No. 29) 5′ GTAGCCTAGCTACCCCTAGGTCTAGGC First-RED RandomAdapter part I (Seq. ID. No. 30) 5′ CTACGATACGATAGGGCCTAAGAGTAG-NNFirst-RED Random Adapter part 2 (Seq. ID. No. 31)5′ CTACTCTTAGGCCCTATCGTATCGTAG

Throughout this application, various publications may have beenreferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which this inventionpertains.

The embodiments described above are given as illustrative examples only.It will be readily appreciated that many deviations may be made from thespecific embodiments disclosed in this specification without departingfrom the invention. Accordingly, the scope of the invention is to bedetermined by the claims below rather than being limited to thespecifically described embodiments above.

1. A probe set composition for the detection of genomic sequences, toprovide terminal signaling per probe and to enable global amplification,global labeling and global addition of reporters to said probe set,wherein said probe set composition comprises a pool of modified cDNAprobes, wherein each probe comprises, as a central target-specificsequence, a single truncated cDNA segment comprising a copy of the 3′end of each mRNA transcript or an amplified set of said probes, whereineach probe further comprises an appended terminal tri-functionalsequence on one or both ends; wherein said sequence comprises at leastone of SEQ ID NO: 1 and its complement, SEQ ID NO: 2 and its complement,SEQ ID NO: 3 and its complement or SEQ ID NO: 4 and its complement, andwherein the tri-functional sequence is: (i) a common universal linkerfor binding a common reporter unit to each probe of the probe set,wherein each reporter unit comprises at least one terminal singlestranded polynucleotide linker complementary to the universal linker ofthe members of the probe set and an attached reporter segment,preferentially comprising double stranded DNA, that additionallycomprises at least two labeling molecules, or (ii) a common universalprimer binding site for globally copying and amplifying the probe set,or (iii) a common universal reporter binding site wherein the universalreporter comprises a single-stranded polynucleotide that iscomplementary to the universal reporter binding site and thatadditionally comprises at least one labeling compound that is directlyattached to the 5′ end or to an internal base of said polynucleotide,and wherein said labeled polynucleotides are incorporated into themembers of the probe set during amplification and/or appended to themembers of the probe set by hybridization.
 2. The probe set of claim 1,wherein each probe of said probe set comprises said terminaltri-functional sequence on both ends.
 3. The probe set of claim 2,wherein said tri-functional sequences at both ends of each probe areselected from the group consisting of SEQ ID Numbers 1-4, and are thecommon primer binding site and wherein all the probes in the probe setcan be amplified with primers having the same sequence.
 4. The probe setof claim 2, wherein each probe in said probe set further comprises apoly-T primer segment in between the central target-specific sequenceand said tri-functional sequence at the 5′ end, wherein said poly-Tsegment is inserted using a poly-T primer composition comprising saidtri-functional sequence to globally copy the mRNA transcripts and arandom sequence in between the central target-specific sequence and saidtri-functional sequence at the 3′ end, wherein the random sequence isinserted by using a random extender composition or a random adaptercomposition to append said tri-functional sequence at the 3′ end of saidcDNA probes and wherein said random extender composition comprises apolynucleotide with a 5′ tri-functional sequence, a 3′ random sequencein the range of about 4 to about 10 randomly variable bases and,optionally, a terminal 3′ end that is chemically blocked to preventpolymerase extension, and wherein said random adapter compositioncomprises two polynucleotides joined together by complementary basesthat comprise said tri-functional sequence; wherein one polynucleotidefurther comprises a short overhang of about 1 to about 6 randomlyvariable bases.
 5. The probe set of claim 1, wherein the probe setcomprises a subset of different probes for each target sequence, whereineach probe of the subset comprises a partial copy of a mRNA transcriptand the terminal tri-functional sequences at one or both ends.
 6. Theprobe set of claim 1, wherein said probe set comprises two or more probesubsets and wherein said probes in each probe subset comprise one orboth of a different tri-functional sequence selected from the groupconsisting of SEQ ID Numbers 1-4, or a different set of labels orreporters from the probes in the other subset(s).
 7. The probe set ofclaim 1, wherein said tri-functional sequence binds a nucleotide linkingsequence of a reporter such that said reporter is selected from thegroup consisting of linear labeled fragments of duplex DNA thatterminate in single stranded universal linkers, and arrays of suchreporters joined end to end.
 8. The probe set of claim 1, wherein saidtri-functional sequence binds a multilinker such that said multi-linkercomprises one or more joined polynucleotides that form a universallinker at one end and a plurality of reporter linkers at the oppositeend.
 9. The probe set of claim 1, wherein said tri-functional sequenceare suitable for chemical cross-linking between bound linkers.
 10. Theprobe set of claim 1, wherein said tri-functional sequence binds aprimer which is labeled such that the probe set is terminally labeled.